WO2008083493A1 - Stabilisation de structures de peptides cycliques - Google Patents

Stabilisation de structures de peptides cycliques Download PDF

Info

Publication number
WO2008083493A1
WO2008083493A1 PCT/CA2008/000048 CA2008000048W WO2008083493A1 WO 2008083493 A1 WO2008083493 A1 WO 2008083493A1 CA 2008000048 W CA2008000048 W CA 2008000048W WO 2008083493 A1 WO2008083493 A1 WO 2008083493A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
intein
cdr
lariat
sequence
Prior art date
Application number
PCT/CA2008/000048
Other languages
English (en)
Other versions
WO2008083493A8 (fr
Inventor
Ronald C. Geyer
Original Assignee
University Of Saskatchewan
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Saskatchewan filed Critical University Of Saskatchewan
Priority to CA002675024A priority Critical patent/CA2675024A1/fr
Priority to US12/522,708 priority patent/US20130273553A9/en
Priority to EP08700513A priority patent/EP2106446A4/fr
Publication of WO2008083493A1 publication Critical patent/WO2008083493A1/fr
Publication of WO2008083493A8 publication Critical patent/WO2008083493A8/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • Trans dominant agents such as small molecules, antisense RNA, ribozymes, RNAi, antibodies, and dominant negative proteins have been developed that make it easier to perform reverse analysis in diploid organisms (Geyer, CR. & Brent, R. (2000) Methods Enzymol. 328:178-208). These agents inactivate gene products without altering the genetic material that encodes them.
  • systematic reverse analysis of protein function requires agents that can be easily and rapidly generated against any given target, that can inhibit protein interactions and activities, and that can block specific interactions with a protein while leaving other interactions unperturbed.
  • reverse analysis must also be performed with reagents that directly inhibit the target rather than blocking steps in transcription or translation of the target.
  • Intracellular inhibitors of protein function with these characteristics can be rapidly obtained by genetically selecting conformational ⁇ constrained, scaffolded peptides (peptide aptamers) from combinatorial peptide aptamer libraries using the yeast two-hybrid assay (Geyer, CR. & Brent, R. (2000) Methods Enzymol. 328:178-208).
  • Constrained peptides are preferred as they generally bind tighter and are more stable (Davidson, A. R. & Sauer, R.T. (1994) Proc. Natl. Acad. Sci. USA 91 :2146-2150) than linear peptides.
  • Combinatorial libraries of peptide aptamers should in principle contain members that bind any target.
  • the scaffold protein enhances solubility and allows a transcription activation domain to be fused to the peptide aptamer, which is essential for the yeast two-hybrid assay.
  • Peptide aptamers are useful for validating proteins as therapeutic targets however displaying peptides on the surface of scaffolds limits their use as drugs or drug-leads as they are usually not membrane permeable and they are susceptible to degradation by proteases.
  • the size of the scaffold protein also prevents the synthesis of peptide aptamers by synthetic peptide chemistry and makes solving their structure difficult.
  • peptides can be constrained by cyclization and there are many examples of natural and synthetic cyclic peptide inhibitors (Horswill, A.R. & Benkovic, SJ. (2005) Cell Cycle 4:552-555). Recently, methods have been developed to express genetically encoded cyclic peptides using engineered inteins (Scott, CP. et al. (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643). Cyclic peptide have advantages over peptide aptamers in that they are resistant to exoproteases and their small size makes them amenable to chemical synthesis, structural studies, and membrane transport.
  • Combinatorial libraries of cyclic peptides have been screened using forward and reverse approaches to isolate cyclic peptides that inhibit cellular processes (Kinsella, T.M. et al. (2002) J. Biol. Chem. 277:37512-37518, Nilsson, L.O. et al. (2005) Protein Pept. Lett. 12:795-799) and disrupt protein interactions (Horswill, A.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101 :15591- 15596), respectively.
  • Antibodies are non-cyclic proteins that have a very well characterized structure made up of a number of domains having a recognizable tertiary structure. Each domain in an antibody molecule has a similar structure of two beta sheets packed tightly against each other in a compressed antiparallel beta barrel. This conserved structure is termed the immunoglobulin fold. The fold is generally stabilized by hydrogen bonding between the beta strands of each sheet, by hydrophobic bonding between residues of opposite sheets in the interior, and by a disulfide bond between the sheets. The folds of variable domains have 9 beta strands arranged in two sheets of 4 and 5 strands. Each variable region is made up from three complementarity determining regions (CDR) separated by four framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the CDR's are the most variable part of the variable regions, and perform the antigen binding function. It has been shown that the function of binding antigens can also be performed by fragments of a whole antibody.
  • Example binding fragments are (i) the Fab fragment consisting of the VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (iv) the dAb fragment (Ward, E. S.
  • One aspect of the invention discloses a genetic assay that may be used to isolate peptide lariats that interact with a target protein using the yeast two-hybrid interaction trap (Gyuris, J. et al. (1993) Cell 75:791-803).
  • a lariat consists of a cyclic peptide or "noose” region with a covale ⁇ tly attached transcription activation domain.
  • the invention provides lariats that are compatible with the yeast two-hybrid system by engineering the intein cyclic peptide producing system (Scott, CP. et al. (1999) Proc. Natl. Acad. Sci.
  • Lariat peptides or cyclic peptides based on the noose sequence can be used to study the function or validate the therapeutic potential of protein targets.
  • the invention exemplifies the feasibility of the foregoing approach by generating inhibitors of the bacterial repressor protein LexA.
  • LexA represents a putative antimicrobial target, which when inhibited should potentiate that activity of cytotoxic antibiotics.
  • LexA is bound by activated RecA it undergoes autoproteolysis and no longer represses genes in its regulon (Lin, L.L & Little, J.W. (1988) Bacteriol. 170:2163-2173).
  • LexA mutants that block autoproteolysis (Walker, G.C. (1984) Microbiol. Rev.
  • vectors comprising a host-operable promoter operably linked to a nucleic acid molecule comprising, in order, an activity domain, a modified C-intein domain, an insert, a modified N-intein domain and a transcription termination sequence.
  • a modified intein lariat library comprising a host- operable promoter operably linked to a nucleic acid molecule comprising, in order, an activity domain, a modified C-intein, an insert having a random peptide or antibody single chain variable fragment (ScFv) encoding oligonucleotides, or a random genomic fragment inserted therein, a modified N-intein and a transcription termination sequence.
  • ScFv refers to an antibody fragment consisting of immunoglobulin variable (V) domains of heavy (H) and light (L) chains held together by a short linker (Tanaka, T. et al. (2003) Nucl. Acids Res. 31 :e23).
  • De novo ScFvs can be constructed that contain specific framework regions from chosen light and heavy chain variable domains and that contain random complementary determining regions.
  • immune and non-immune ScFv libraries can be generated using RT-PCR to amplify light and heavy chain variable domains from total RNA purified from B lymphocytes of peripheral blood.
  • the immune libraries can be generated from animals challenged with a specific antigen or from animals with a specific disease. Genomic fragments refer to randomly or rationally generated fragments of DNA derived from genomic DNA or cDNA.
  • methods for identifying a cyc ⁇ c-like peptide, ScFv, or genomic fragment that interacts with a target molecule. These methods may for example take place inside of an host organisms comprising: (i) transforming the modified intein library as described above into a suitable host or host ceils or cell line; (ii) transforming said with a nucleic acid molecule encoding the target molecule attached to the second activity domain arranged for expression in said host; (iii) identifying host cells comprising a detectable product generated by bringing together the activity domains through an interaction between a member of the intein library and the target molecule; and (iv) recovering the library member from the host cell expressing the detectable product and sequencing the random peptide, ScFv or genomic fragment encoding oligonucleotide.
  • RNA encoding a protein has been developed to couple the RNA encoding a protein to the expressed protein including ribosome display (Mattheakis, et al., (1994) Proc. Nat. Acad. Sci. USA 91 :9022) and mRNA display (Roberts, R.W. & Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA 94:12297). Any of these assays or similar assays not listed that couple the DNA or RNA encoding nucleic acid to its expressed protein can be used to isolate cyclic-like peptides, genomic fragments or ScFvs that interact with a protein target.
  • the invention also provides cyclical peptides, ScFv, or genomic fragment isolated as described above.
  • the present invention provides methods that may be used to generate cyclic and lariat peptide inhibitors of selected targets, which can be used for a variety of purposes.
  • the cyclic peptides can be used as drugs to inhibit disease-causing targets. They can also be used as affinity reagents for validating the therapeutic potential of targets or in general applications that require affinity reagents.
  • the lariat peptides are useful for applications that use cyclic peptides, but may also require a tag (tail) to be covalently attached to the cyclic peptide. These tags can encode yeast two hybrid transcription activation domains as described herein.
  • the tags may also encode moieties required for other protein interaction detection systems including: split-ubiquitin system (Stagljar et al., (1998) Proc. Natl. Acad, Sci. USA 95:5187), protein-fragment complementation assay (Remy I. & Michnick S.W. (1999) Proc. Natl. Acad. Sci. USA 96:5394), repressor reconstitution assay (Hirst et al., (2001) Proc. Natl. Acad. Sci. USA 98:8726), SOS recruitment system (Broder et al., (1998) Curr. Boil. 8:1121), phage display (Smith, G.P.
  • the tags may also encode labels for labelling targets (fluorescence, radioactivity etc), localization sequences, membrane permeation sequences, antibody epitope tags, nucleic acid sequences to detect and quantify the amount of bound target, or small molecules. Other suitable uses will of course be apparent to one of skill in the art.
  • libraries of lariat peptide can be generated in a variety of organisms.
  • Specific lariat peptides in these libraries that interact with a specific target can be genetically selected using the protein interaction assays described above.
  • the yeast two- hybrid assay has many advantages including but by no means limited to the following.
  • Cyclic peptides are relatively stable and small, increasing their in vivo stability and cellular permeability.
  • the invention provides peptides that may be adapted for intracellular peptide delivery.
  • manipulation of the HIV-1 -derived Tat- peptide system has been utilized for intracellular peptide delivery. See for e.g. Caron et al. (2001) Intracellular delivery of a Tat-eGFP fusion protein into muscle cells. MoI. Therap. 3(3): 310-18; Wadia and Dowdy (2003) Modulation of cellular function by TAT mediated transduction of full length proteins; and EP 656950 B1.
  • the penetratin, transportan, and MAP (KLAL) peptides can be used to mediate intracellular delivery. See for e.g. Hallbrink et al. (2001) Cargo delivery kinetics of cell-penetrating peptides. Biochim. Biophys. Acta. 1515(2): 101-09; Thore ⁇ et al. Uptake of analogs of penetratin, Tat(48-60) and oligoarginine in live cells. Biochem. Biophys. Res. Commun. 307(1): 100-07; WO 2006/101283 A1 ; and Howl et al.
  • oligoarginine fusion proteins can be delivered intracellular ⁇ . See for e.g. Han et al. (2001) Efficient intracellular delivery of exogenous protein GFP with genetically fused basic oligopeptides. MoI. Cells. 12(2): 267-71; Futaki et al. (2001) Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem.
  • myristoylated peptides can be delivered intracellular ⁇ . See for e.g. Nelson et al. (2007) Myristoyl-based transport of peptides into living cells. Biochemistry 46(51): 14771-81; and EP 651805 B1.
  • the yeast two-hybrid assay is an easy, fast, and automatable assay.
  • the yeast two-hybrid system can be performed in array format. This allows arrays of lariat peptides to be generated. These arrays can be used to rapidly generate lariat peptides against specific targets using automated robotics.
  • the patterns of lariat peptides that interact with different targets can be used to characterize targets. For example, targets with similar binding surfaces should interact with similar lariat peptides in the array.
  • lariat peptides can be used to pull down target complexes to identify interaction partners.
  • lariats can be immobilized onto surfaces creating protein micro-array chips to detect protein levels.
  • additional functional domains may be attached to the lariat including visualization, and destruction domains.
  • the invention provides recombinant nucleic acid sequences encoding a split intein polypeptide.
  • the split intein polypeptide may include, in amino to carboxy order: an l c domain comprising an F block and a G block, the F block being at least 80% identical to the sequence rVYDLpV ** a - - HNFh, designated respectively as positions F1 to F16, and the G block being at least 80% identical to the sequence NGhhhHNp, designated respectively as positions G1 to G8; an extein domain attached to the C terminal portion of the G block; and, an I N domain attached to the C terminal portion of the extein domain, the I N domain comprising an A block and a B block, the A block being at least 80% identical to the sequence Ch - - Dp - hhh - - G, designated respectively as positions A1 to A13, and the B block being at least 80% identical to the sequence G - - h - hT - - -
  • a capital letter represents an amino acid designated by the single letter amino acid code
  • "h” represents a hydrophobic residue selected from the group consisting of G, B, L, I, A and M
  • a represents an acidic residue selected from the group consisting of D and E
  • "r u represents an aromatic residue selected from the group consisting of F, Y and W
  • "p” represents a polar residue selected from the group consisting of S, T and C
  • "-" represents any amino acid
  • * " represents optional gaps.
  • the residue encoded at position G7 is Q, W, F, L, I, Y, M, V, R, K, H, E or D; and/or
  • the residue encoded at position G6 is L, N, D, W, F, I, M or Y; and/or (c) the residue encoded at position B11 is K, Y, F 1 W, H, Q or E; and/or
  • the residue encoded at position F13 is F 1 L or I; and/or, (h) the residue encoded at position F14 is W, F, Y, L, K or R; and/or (i) the residue encoded at position F15 is W or L; and/or, (j) the residue at position B9 is not R or T and is a non-catalytic amino acid for an N-X acyl shift; and/or,
  • the residue at position B10 is not R or T and is a non-catalytic amino acid for an N-X acyl shift; and/or, (I) the residue at position F2 is not R or T and is a non-catalytic amino acid for an N-X acyl shift; and/or, (m)the residue at position F6 is not S, T or C and is a non-catalytic amino acid for a transesterification reaction involving a nucleophilic amino acid at position G8 attacking an ester or thioester bond.
  • the extein domain may include an immunoglobulin encoding region that encodes an immunoglobulin molecule comprised of a heavy chain variable region attached by linkers to a light chain variable region, a first linker attaching the C-terminal region of the heavy chain variable region to the N-terminal region of the light chain variable region and a second linker attaching the N-terminal region of the heavy chain variable region to the C-terminal region of the light chain variable region, wherein the linkers comprise a polypeptide chain of at least 10 amino acids (or an integer number of amino acids between 10 and 50).
  • the heavy chain variable region may include one or more heavy chain framework regions selected from the group consisting of HFR1 , HFR2, HFR3, and HFR4; and the heavy chain variable region further comprises one or more complementarity determining regions selected from the group consisting of CDR-M, CDR-H2, CDR-H3; with the heavy chain framework and complementarity determining regions arranged in accordance with the formula HFR1- CDR-H1-HFR2--CDR-H2-HFR3-CDR-H3--HFR4.
  • the light chain variable region may include one or more light chain framework regions selected from the group consisting of LFR1, LFR2, LFR3 and LFR4; and the light chain variable region further comprises one or more complementarity determining regions selected from the group consisting of CDR-L1 , CDR-L2 and CDR-L3; with the light chain framework and complementarity determining regions arranged in accordance with the formula LFR1--CDR-L1--LFR27-CDR-L2--LFR3-- CDR-L3--LFR4. In these structural formulae:
  • HFR1 is a first heavy chain framework region consisting of a sequence of about 30 amino acid residues (or any integer value or range therein from 20 to 40);
  • HFR2 is a second heavy chain framework region consisting of a sequence of about 14 amino acid residues (or any integer value or range therein from 10 to 30);
  • HFR3 is a third heavy chain framework region consisting of a sequence of about 29 to about 32 amino acid residues (or any integer value or range therein from 20 to 50);
  • HFR4 is a fourth heavy chain framework region consisting of a sequence of 7 to about 9 amino acid residues (or any integer value or range therein from 5 to 15);
  • CDR-H1 is a first heavy chain complementary determining region (which may form example be any integer value or range therein from 10 to 100 amino acids is length);
  • CDR-H2 is a second heavy chain complementary determining region (which may form example be any integer value or range therein from 10 to 100 amino acids is length);
  • CDR-H3 is a third heavy chain complementary determining region
  • LFR1 is a first light chain framework region consisting of a sequence of about 22 to about 23 amino acid residues (or any integer value or range therein from 15 to 35);
  • LFR2 is a second light chain framework region consisting of a sequence of about 13 to about 16 amino acid resid ⁇ es (or any integer value or range therein from 15 to 35);
  • (x) LFR3 is a third light chain framework region consisting of a sequence of about 32 amino acid residues (or any integer value or range therein from 20 to 40);
  • LFR4 is a fourth light chain framework region consisting of a sequence of about 12 to about 13 amino acid residues (or any integer value or range therein from 5 to 25);
  • CDR-L1 is a first light chain complementary determining region (which may form example be any integer value from 10 to 100 amino acids is length);
  • CDR-L2 is a second light chain complementary determining region (which may form example be any integer value from 10 to 10O amino acids is length);
  • CDR-L3 is a third light chain complementary determining region (which may form example be any integer value from 10 to 100 amino acids is length).
  • the invention further provides host cells that include the foregoing recombinant nucleic acids, including cells in which the split intein polypeptide is processed in the host cell in a self catalyzed reaction to form at least one cyclized polypeptide having no more than one linear terminal end (such as an immunoglobulin molecule having no more than one linear terminal end and having the conformation of an immunoglobulin fold).
  • the cyclized polypeptide may have one linear terminal end, being a C-terminal end or an N-terminal end, such as a lariat peptide (which may include a lactone or lactam junction).
  • the cyclized polypeptide may be cyclic, so that it has no linear terminal end.
  • a host cells of the invention may be adapted for use in methods for assaying interactions between fusion proteins.
  • cells of the invention may include: a first recombinant gene coding for a prey fusion protein, the prey fusion protein comprising a transcriptional repressor or activator domain and a first heterologous amino acid sequence; a second recombinant gene coding for a bait fusion protein, the bait fusion protein comprising a DNA-binding domain and a second heterologous amino acid sequence; and, a recombinant reporter gene coding for a detectable gene product, the recombinant reporter gene comprising an operator DNA sequence capable of binding to the DNA binding domain of the bait fusion protein; wherein expression of the reporter gene is modulated in response to binding between the first heterologous amino acid sequence and the second heterologous amino acid sequence; and, wherein at least one of the recombinant genes comprises the foregoing recombinante nucleic acids.
  • the invention provides immunoglobulin molecules having no more than one linear terminal end, including molecules having the conformation of an immunoglobulin fold comprised of a heavy chain variable region attached by linkers to a light chain variable region.
  • a first linker may be present attaching the immunoglobulin molecules having no more than one linear terminal end, including molecules having the conformation of an immunoglobulin fold comprised of a heavy chain variable region attached by linkers to a light chain variable region.
  • a first linker may be present attaching the
  • linkers may be flexible covalent molecular links of at least approximately 50 Angstroms in length, such as polypeptide chains of about 15 amino acids in length, or from 14 to 25 amino acids in length (for example made up of glycine and serine residues).
  • FIG. 1 lntein Catalyzed Protein Splicing Reactions, (a) Self-splicing intein reaction, lntein domains (black) catalyze a self-splicing reaction that results in the joining of the extein domains (white), (b) Split-int ⁇ in reaction.
  • the intein is split into two separate proteins. One protein contains the N-Extein and N-lntein and the other protein contains the C-lntein and C-Extein. Interaction between the intein domains results in joining of the extein domains, (c) Split-lntein protein cyclization reaction.
  • the intein domains are swapped relative to the extein domains. Intein domains fold together and catalyze cyclization of the extein domain.
  • FIG. 1 Schematic of the Intei ⁇ -M ⁇ diated Peptide Cyclization Reaction.
  • Step 1 intein folding - C-intein and N-intein domains interact to form a catalytically active intein structure.
  • Step 2 N-intein cleavage - Intein catalyzes the cyclization of extein and the cleavage of the N-intein domain.
  • Step 3 C-intein release - C-intein domain is cleaved resulting in the formation of the cyclic peptide.
  • FIG. 3 Formation of Lariat Intein.
  • the lariat intein is an intermediate product in the intein-catalyzed cyclization reaction.
  • the C-terminal amino acid in the lariat peptide is covalently attached through a lactone bond to a specific nucleophilic residue in the C- lntein domain (l c )-
  • the cyclized section of the lariat intein "noose” is used to display (i) random peptides, (ii) genomic fragments, and (iii) antibody single chain variable fragments (ScFv).
  • the l c domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), haemagglutinin tag (HA).
  • NLS nuclear localization sequence
  • ACT transcription activation domain
  • HA haemagglutinin tag
  • FIG. 4 Formation of Unprocessed Intein.
  • the unprocessed intein is formed by blocking Step (i) in the intein-catalyzed cyclization reaction,
  • the extein or region constrained between the C-lntein (l c ) and N-lntein (I N ) domains is used to display (i) random peptides, (ii) genomic fragments, and (iii) antibody single chain variable fragments (ScFv).
  • the Ic domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), and haernagglutinin tag (HA).
  • NLS nuclear localization sequence
  • ACT transcription activation domain
  • HA haernagglutinin tag
  • the dicysteine intein is formed by blocking Step (i) in the intein-catalyzed cyclization reaction.
  • the dicysteine intein contains one Cys after the C-lntein domain (Ic) and one Cys at in the first amino acid position of the
  • N-lntein domain (I N )- (b)
  • the extein, or region constrained between the two Cys amino acids is used to display (i) random peptides, (ii) genomic fragments, and (iii) antibody single chain variable fragments (ScFv).
  • the Ic domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), and haernagglutinin tag (HA).
  • Figure 6 Conversion of Lariat, Unprocessed, and Dicysteine Intein to Cyclic and Linear Peptides
  • the lactone-cyclized peptide or protein in the lariat intein can be converted to a head to tail cyclized peptide or protein or a linear peptide or protein
  • the constrained peptide or protein in the unprocessed intein can be converted to a head to tail cyclized peptide or protein or a linear peptide or protein
  • the constrained peptide or protein in the dicysteine intein can be converted to a Cys cross-linked or disulfide bond cyclized peptide or protein or a linear peptide or protein.
  • I N is the N-lntein domain and l c is the C-lntein domain.
  • the l c domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), and haernagglutinin tag (HA).
  • NLS nuclear localization sequence
  • ACT transcription activation domain
  • HA haernagglutinin tag
  • FIG. 7 Yeast Two-Hybrid Assay Using the Lariat, Unprocessed, and Dicysteine inteins.
  • (a) Combinatorial lariat intein libraries are screened using the yeast two-hybrid assay.
  • the lariat intein contains a transcription activation domain (ACT) fused to the Ic domain, which is required to activate the reporter genes in the yeast two-hybrid assay
  • ACT transcription activation domain
  • the unprocessed intein contains a transcription activation domain (ACT) fused to the Ic domain, which is required to activate the reporter genes in the yeast two-hybrid assay
  • (c) Combinatorial dicysteine intein libraries are screened using the yeast two-hybrid assay.
  • the dicysteine intein contains a transcription activation domain (ACT) fused to the ! c domain, which is required to activate the reporter genes in the yeast two-hybrid assay.
  • the I 0 domain is also shown fused to a nuclear localization sequence (NLS), and haemagglutinin tag (HA).
  • the C-lntein domain (I 0 ) has conserved blocks F and G, and the N-lntein (I N ) domain has conserved blocks A, and B.
  • conserved amino acids are numbered according to their block letter and position number.
  • An enlargement of the splice site at the Ic-Extein-I N boundaries is shown.
  • the I 0 intein is numbered from C-ternninus to N-terminus using the labelling scheme lc-i, lc-2» lc-3, ⁇ •» or according to block letter and position number.
  • Block G is number 1-8 and Block F is numbered 1-16.
  • the I N intein is numbered from the N-terminus to the C-terminus using the labelling scheme I N+1 , I N+2 . I N+3 ⁇ ••, or according to block letter and position number.
  • Block A is numbered 1-13 and Block B is numbered 1-14.
  • the extein is numbered from N- terminus to C-terminus using the labelling scheme lc + i, !c + 2, lc + s •--. or from C-terminus to N-terminus using the labelling scheme l N -i, IN-2, I N - S • ⁇ --
  • the consensus sequence for each block is shown below the block.
  • Step (iv): Ester to amide shift - The ester or thioester bond is converted to an amide bond by the thermodynamically favoured X to N acyl shift. Definitions: X O or S depending on Ser or Cys. I N is the N-lntein domain and Ic is the C-lntein domain.
  • X S or O depending on Cys, or Ser/Thr.
  • I N is the N-lntein domain and I 0 is the C-lntein domain.
  • the loi (G8) nucleophile at the Ic-Extein junction undergoes a nucleophilic attack on the ester or thioester formed in Step (i) and produces the branched intermediate.
  • Step (iv): Lactone to Lactam Shift - The lactone cyclized intein is converted to the lactam by the thermodynamically favoured X to N acyl shift. Definitions: X S or O depending on Cys, or Ser/Thr. I N is the N-lntein domain and l c is the C-lntein domain.
  • Step (ii) Transesterification reaction). If only Step (ii) is blocked then the unprocessed intein can undergo two side reactions.
  • Side reaction (iii) (Asn cyclization) causes the Ic domain to be cleaved from the unprocessed intein.
  • Side reaction (iv) (Ester hydrolysis) cause cleavage of the I N domain from the unprocessed intein.
  • Steps (i) (N-X acyl shift) and (iii) (Asn cyclization) need to be inhibited.
  • the l c domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), and haemagglutinin tag (HA).
  • X S or O depending on Cys, or Ser/Thr.
  • Step (a) The dicysteine intein is generated by inhibiting Step (ii) (Transesterification reaction). If only Step (ii) is blocked then the unprocessed intein can undergo two side reactions.
  • Side reaction (iii) (Asn cyclization) causes the Ic domain to be cleaved from the unprocessed intein.
  • Side reaction (iv) (Ester hydrolysis) cause cleavage of the I N domain from the unprocessed intein.
  • Steps (i) N-X acyl shift) and (iii) (Asn cyclization) need to be inhibited.
  • the Ic domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), and haemagglutinin tag (HA).
  • X S or O depending
  • FIG. 14 Generation of the Lariat Intein.
  • Step (iii) (Asn cyclization) needs to be blocked.
  • the lariat intein can undergo the side reaction (iv) (Lactone hydrolysis).
  • Step (iv) (Lactone hydrolysis) should be reduced.
  • the l c domain is shown fused to a nuclear localization sequence (NLS), transcription activation domain (ACT), and haemagglutinin tag (HA).
  • X S or O depending on Cys, or Ser/Thr.
  • FIG. 15 Isolation of Anti-L ⁇ xA lariats, (a) Intein-mediated peptide cyclizatio ⁇ . (i) Unprocessed intein undergoes an N-to-S acyl shift using the I N+1 cysteine at the peptide- ⁇ junction, (ii) Transesterification reaction involving leu serine at the l c -peptide junction and the thioester formed in step (i), which releases the I N domain producing the lariat intermediate, (iii) In the intein producing cyclic peptide system, l c+2 asparagine undergoes a side chain cyclization, which releases the l c domain and generates a lactone-cyclized peptide that undergoes a thermody ⁇ amically favoured O to N acyl shift to produce a lactam-cyclized peptide.
  • Lariat intein contains an asparagine to alanine mutation at position l c .i, which blocks the asparagine side chain cyclization reaction.
  • Inactive intein contains the same mutations as the lariat intein and a serine to alanine mutations at position l c+ i and a cysteine to alanine mutation at position I N+1 .
  • Cysteine to alanine mutation at l N+ i blocks the N to S acyl shift.
  • Serine to alanine mutation at I 0+I blocks the transesterification reaction.
  • X represents amino acids coded by the NNK codon.
  • the lariat contains a transcription activation domain, which is used to select anti-LexA lariats using the yeast two-hybrid interaction trap, (d) Amino acid sequences of the noose region from two anti-LexA lariat peptides (L1 and L2). Amino acids from the combinatorial region are bolded and dashes are used to align common amino acids in L1 and L2.
  • FIG. 16 Analysis of Combinatorial Lariat Library- Sequences from seventeen lariat library plasmids (pi L-XX). Bold amino acids are constant. * represent stop codons. X represents amino acids coded by the NNK codon. 35% of the library contains random seven amino acids peptides with no stop codons.
  • FIG. 17 Analysis of L2 Lariat
  • plL-L2 and plN-L2 are designed to produce lariat and unprocessed inteins, respectively.
  • the unprocessed intein is at - 23 kDa and the lariat is at - 9 kDa.
  • plL-01 is a lariat expression plasmid with a CGPC peptide noose.
  • plL-L2 is a lariat expression plasmid with an L2 noose.
  • plN-L2 is a mutant lariat expression plasmid with l_2 noose that produces only the unprocessed intein.
  • Yeast growth on nonselective His ' ,Trp " glucose media
  • Yeast grown on His " Trp ' Leu ' Ade Xgal galactos ⁇ /sucrose media that selects for the activation of LEU2, ADE2, and LacZ yeast two-hybrid reporter genes.
  • L2 peptide was immobilized onto a CM5 sensor chip and LexA (11 ⁇ M - 110 ⁇ M) was passed over the sensor chip.
  • LexA 11 ⁇ M - 110 ⁇ M
  • the response curve of each point was used to determine the dissociation constant (K,,) using the BiaEvaluation (Biacore) fitting software. Standard deviation was calculated using the different LexA concentrations.
  • FIG. 19 Mechanism for Lariat Cleavage By NaOH. Hydrolysis of the lariat lactone by Na 18 OH can occur by two mechanisms, (a) The first mechanism involves the hydrolysis of the ester bond causing 18 O incorporation at the tyrosine carboxylic acid at position (?N- I )- (b) The second mechanism involves an ⁇ -H elimination to generate dihydroalanine, followed by a Michael addition, which incorporates the O 18 at the serine side chain at position (I 0+1 ).
  • FIG. 20 Quantification of Lariat Prior to MS analysis,
  • the 16 O product has a calculated mass of 966.420 m/z and the 18 O product has a calculated mass of 968.420 m/z.
  • Trypsin digest of the Na 18 OH treated lariat produces a peptide fragment containing serine at position (lc + i) (IFDIGLPQDHNFLLANGAIAHASR).
  • the mass of this fragment is 2590.352 m/z corresponding to a product 1 Da heavier than the predicted 16 O incorporated product.
  • a 1 Da shift can be attributed to deamidation of asparagine.
  • the asparagine at position (l c . 7 ) is susceptible to base-catalyzed deamidation as it is N- terminal to a glycine (7-10). The 2+, 3+ 4+ and 5+ charged fragments were analyzed and similar results were obtained.
  • Figure 21 Biological Activity of L2 Lariat and Cyclic Peptide, (a) Inhibition of MMC- induced LexA cleavage by L2 lariat.
  • Cell extracts were analyzed by Western analysis using Anti-LexA antibody at 0, 1 , 2, and 3 hours after MMC addition, (b) Inhibition of MMC-induced expression of sulA-GFP in SMR6039-DE3.
  • Normalized percent cell survival is calculated by dividing the number of colony forming units (cfu) after one hour by the number of cfu at the zero hour time point.
  • the uninduced control or the no peptide control is normalized to 100 %. Error bars represent the standard deviation of three independent experiments.
  • Figure 22 Linear L2 Peptide Inhibits Cell Survival and Potentiates Mitomycin C Activity. Survival assay for BL21 -CP cells treated synthetic linear L2 peptide. Cell survival is reported relative to the untreated control. Error bars represent the standard deviation of three independent experiments.
  • Figure 24 The affect of lariat mutations on lariat stability and processing.
  • Three positions in the i ⁇ tein construct, G6, G7, and B11 were mutated to amino acids listed.
  • the wild-type intein process all steps in the intein reaction and produces a cyclic peptide.
  • the G6: His, G7: Ala, and B11 : Arg is the lariat producing intein construct.
  • (-) indicates the lariat formation and processing was not characterized.
  • % lariat is the amount to unhydrolyzed lariat.
  • % processing is the amount of undergoing the first two steps in the lariat reaction.
  • FIG. 25 Amino Acid Positions In The Diversified Complementarity Regions (CDRs) Of The ScFv Libraries.
  • CDRs Complementarity Regions
  • the names of the CDRs are listed above the tables and the positions are labelled with numbers corresponding to the Kabat database. The letters under the numbers refer to the amino acids in that position in the single letter amino acid code.
  • X denotes a variable position.
  • Figure 26 Time course analysis of ScFv lariat processing.
  • the unprocessed intein is at ⁇ 54 kDa and the lariat is at ⁇ 42 kDa.
  • FIG. 27 Yeast two-hybrid comparison of ScFv library interactions. K4, cyc-K4, T4 and cyc-T4 libraries were screened against a pool of five baits: Bcr-Abl SH2 Domain, Bcr-
  • Head to tail peptide cyclization resulting in a continuous amide peptide backbone, has been successfully used to constrain and stabilize peptides and to improve their biological activity.
  • a variety of in vitro chemical and enzymatic strategies for cyclizing peptides from their linear precursors have been developed.
  • Recently, methods using inteins have been developed to synthesize head to tail cyclic peptides in vivo (Scott, CP. et al. (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643).
  • Inteins are self-splicing proteins that are present in between exteins in a precursor protein. Inteins remove themselves from the precursor protein, resulting in a joining of the exteins (Fig. 1a). Naturally occurring and engineered inteins and split inteins ligate proteins and peptides together (Fig. 1b). Based on these results, inteins have been further engineered to generate cyclic proteins and peptides. To do this, the order of the intein domains are changed (FIg. 1c) to enable the head to tail cyclization of the extein domain.
  • head to tail cyclized peptides that disrupt specific protein-protein interaction using a genetic selection strategy in bacteria (Horswill, A.R. et al. (2004) Proc. Natl. Acad. Sci. USA 101:15591-15596, Tavassoli, A. & Benkovic, SJ. (2005) Agnew Chem. Int. Ed. Engl. 44:2760-2763).
  • head to tail cyclized peptides lack free N-terminal or C-terminal ends which means that reporters or activators cannot be attached thereto.
  • the present invention describes the construction and application of the "lariat" intein or lariat precursors (unprocessed or dicysteine inteins) in the yeast two-hybrid assay or other selection technologies described above and/or known in the art.
  • the lariat is a new peptide construct that has no C-terminus and represents a novel class of cyclic peptides.
  • Lariat inteins are generated by modifying the in vivo intein-mediated protein ligation reaction ⁇ Fig. 2).
  • the C-terminus of the lariat intein is looped back and linked to a specific serine in the interior of the peptide via a lactone bond (Fig. 3).
  • Fig. 3 Libraries of random peptides, ScFvs, or genomic fragments can be displayed in the cyclic or noose region of the lariat (Fig. 3).
  • the lariat unlike a head to tail cyclized peptide, has a free N-terminus that allows the attachment of useful activity domains such as a transcription activation domain, which is necessary for yeast-two hybrid assays.
  • the unprocessed intein (Fig. 4) is an intein construct that is unable to undergo any steps in the intein mediated cyclization. Random peptides, ScFvs, or genomic fragments can be displayed and constrained between the C- and N-intein domains (Fig. 4).
  • the unprocessed intein has a free N- and C-terminus that allows the attachment of useful activity domains at either end such as a transcription activation domain, which is necessary for yeast-two hybrid assays.
  • the dicysteine intein (Fig. 5) is an intein construct that is unable to undergo any steps in the intein mediated cyclization. Random peptides, ScFvs, or genomic fragments can be displayed and constrained between the C- and N-intein domains (Fig. 5). In the dicysteine intein, random peptides, ScFvs, or genomic fragments are flanked by a Cys at each end.
  • the dicysteine intein has a free N- and C-terminus that allows the attachment of useful activity domains at either end such as a transcription activation domain, which is necessary for yeast-two hybrid assays.
  • the lariat construct or the unprocessed intein and dicysteine intein can be used to display libraries of combinatorial cyclic peptides, ScFvs, or genomic fragments fused to a transcription activation domain.
  • ScFv refers to an antibody fragment consisting of immunoglobulin variable (V) domains of heavy (H) and light (L) chains held together by a short linker (Tanaka, T. et a). (2003) Nucl. Acids Res. 31 :e23).
  • the ScFv can be constructed with the V H domain fused to the Ic domain followed by a linker and the V L domain.
  • the V L domain can be fused to the l c domain followed by a linker and the V H domain.
  • ScFv refers to either construct.
  • genomic fragments refer to randomly or rationally generated fragments of DNA derived from genomic DNA or cDNA that are expressed in the lariat, unprocessed intein or dicysteine intein constructs.
  • the yeast two- hybrid assay or other selection technologies can be used to genetically select lariat peptides, unprocessed inteins, and dicyst ⁇ ine inteins that bind to specific targets.
  • RNA, and DNA interactions within cells can also be used to select lariat peptides, unprocessed inteins, and dicysteine inteins that bind to specific targets including two-hybrid systems (reviewed in Vidal M. & Legrain P. (1999) Nucl. Acid Res. 27:919), split-ubiquitin system (Stagljar et al., (1998) Proc. Natl. Acad. Sci. USA 95:5187), protein-fragment complementation assay (Remy I. & Michnick S.W. (1999) Proc. Natl. Acad. Sci.
  • RNA encoding a protein includes ribosome display (Mattheakis, et al., (1994) Proc. Nat. Acad. Sci. USA 91 :9022) and mRNA display (Roberts, R.W. & Szostak, J.W. (1997) Proc. Natl. Acad. Sci.
  • any of these assays or similar assays not listed that couple the DNA or RNA encoding nucleic acid to its expressed protein can be used to isolate cyclic-like peptides, genomic fragments or ScFvs that interact with a target.
  • the lariat and unprocessed inteins that bind specific targets can be used as templates for synthesizing linear or cyclic peptides, ScFvs or genomic fragments that interact with the same target but do not contain any intein sequence (Fig. 6a, b).
  • Dicysteine inteins that interact with a target can be used to design constrained peptides, ScFvs or genomic fragments that interact with the target, but that do not contain any of the intein sequence.
  • the peptide, ScFv, or genomic fragment that are displayed between the C- and N-intein domains are synthesized with flanking Cys.
  • the flanking Cys are used to crosslink and constrain the peptide, ScFv, or genomic fragment (Rg.
  • the dicysteine intein can also be constructed using other cross-linkable amino acids in place of the two- cysteine residues.
  • cross-linkable moieties present on amino acids include but are not limited to: amine-thiol, amine-amine, amine-carboxylic acid, carboxylic acid- carboxylic acid, etc.
  • Further amino acids can be post-translationally modified to incorporate cross-linkable moieties that are not naturally present on amino acids.
  • the cross-linking molecules can be designed such that additional molecules with unique functions can be appended to the peptide, ScFv 1 or genomic fragment. These molecules may include fluorescent labels, localization sequences, purification tags, molecule destruction moieties, etc.
  • 'intein 1 refers to a well-known group of 'splicing proteins'. As discussed herein, a variety of inteins can be modified as discussed below for use in the invention. The N-intein domain and C-intei ⁇ domain from different inteins can also be mixed to create functional inteins.
  • Examples include but are not limited to naturally occurring split-inteins, for example, Aha DnaE-c and Aha DnaE-n, Aov DnaE-c and Aov DnaE-n, Asp DnaE-c and Asp DnaE-n, Ava D ⁇ aE-c and Ava DnaE-n, Cwa DnaE-c and Cwa DnaE-n, Dra Snf2-c and Dra Snf2-n, Npu DnaE-c and Npu DnaE-n, Nsp DnaE-c and Nsp DnaE-n, Oli DnaE-c and Oli DnaE-n, Ssp DnaE-c and Ssp DnaE-n, TeI DnaE-c and TeI DnaE-n, Ter DnaE-3c and Ter DnaE-3n and Tvu DnaE-c and Tvu DnaE-
  • Suitable inteins include those peptides identified as being an intein, that is, a peptide that meets the following criteria ⁇ from a New England Biolabs Webpage):
  • the invention provides methods for isolating lariat inteins, unprocessed inteins, and dicysteine inteins that recognize a selected target, for example using the yeast two-hybrid interaction trap (Fig. 7).
  • Lariat inteins are cyclized peptides, genomic fragments, or ScFvs that have a peptide tag covalently attached to the cyclized or noose region.
  • Lariat peptides are generated by mutating the cyclic-peptide generating intein such that it only undergoes the first two steps in the cyclization reaction.
  • a lariat is an intermediate product in the intein-mediated cyclic peptide reaction.
  • the lariat product contains a tail (for the yeast two-hybrid assay, this is a transcription activation domain) covalently attached through an amide bond to a lactone-cyclized peptide.
  • the lariat peptides are necessary for the yeast two-hybrid assay as this assay requires a transcription activation domain be attached to the cyclic peptide to activate the reporter gene.
  • the yeast two-hybrid assay can be to generate cyclic and lariat peptide affinity agents against a given target.
  • Other activations domains may be utilized, for example, repression domains, split ubiquitin and other two hybrid fusions known in the art, as discussed herein.
  • the first precursor protein referred to as the unprocessed intein
  • the unprocessed intein contains mutations that do not allow any steps to occur in the cyclization reaction.
  • the combinatorial peptide, genomic fragment, or ScFv is constrained by inserting it between C-intein and N-intein domains.
  • the activity domain can be attached to either the C-intein or N-intein domain.
  • the second precursor protein referred to as the dicysteine intein, contains combinatorial peptides, genomic fragments, or ScFvs flanked by cysteines at each end.
  • the dicysteine intein also contains mutations that do not allow the steps in the cyclization reaction to proceed.
  • Combinatorial peptides, ScFvs, or genomic fragments inserted between the C-intein and N-intein domains can be selected that interact with a target molecule.
  • the unprocessed intein or dicysteine intein can be used as affinity agents against a given target.
  • Cyclic peptides based on the sequence of the peptide, ScFv or genomic fragment insert can also be used as affinity agents against a given target.
  • the cysteines at each end of the peptide insert in the dicysteine intein can also be used to cyclize peptide, genomic fragment, or ScFv inserts either through the formation of a disulfide bond or by cross linking the cysteines through a thiol reactive cross linker.
  • Cyclic peptides are utilized in nature to produce high-affinity drug-like effectors. Both naturally occurring and synthetically designed cyclic peptides have been successfully employed as drugs to treat human diseases (Horswill, A.R. & Benkovic, SJ. (2005) Cell Cycle 4:552-555). Cyclic peptides have an advantage for use as drugs since they have diminished proteolytic susceptibility relative to linear peptides (Humphrey, J. M. & Chamberiin, A.R. (1997) Chem. Rev. 97:2243-2266) and they display enhanced binding to their target due to their restricted conformational space (Horton, D.A. ⁇ t al. (2002) J. Comput. Aided MoI. Des.
  • the invention provides a modified intein library and a method of using the library to screen for cyclic peptides, genomic fragments, and ScFvs, which interact with a specific target or interfere with a specific process.
  • the 'specific process' may be protein-protein interactions.
  • a vector which comprises a host-operable promoter operably linked to a nucleic acid molecule comprising, in order, an activity domain, a modified C-intein, an insert, a modified N-intein and a transcription termination sequence.
  • a vector which comprises a host-operable promoter operabiy linked to a nucleic acid molecule comprising, in order, a modified C-intein, an insert, a modified N-intein, an activity domain and a transcription termination sequence.
  • the host-operable promoter is a suitable promoter active in the host that is operably linked to the intein library as described herein for driving expression in the host.
  • E ⁇ xamples of such promoters and termination sequences are well-known in the art as are the hosts in which these elements are functional.
  • a strong viral promoter for example, SV40 or CAMV, may be used.
  • SV40 or CAMV a strong viral promoter
  • one advantage of these constructs is that they would be functional in multiple hosts.
  • a tissue-specific promoter or inducible promoter may be used.
  • the host-operable promoter in the vector is a cassette that can be easily replaced using common molecular biology techniques for inserting different expression cassettes or promoter cassettes upstream of the nucleic acid sequence.
  • the activity domain is selected based on its ability to form a detectable product when in close proximity to a second activity domain.
  • the second activity domain is fused to the target molecule so that interaction between the cyclic peptide, ScFv or genomic fragment encoded by the intein library and the target molecule brings together the two activity domains to produce a detectable product.
  • Suitable activity domains include but are by no means limited to DNA binding domains, transcription activation domains, repression domains, fluorescent proteins and localization sequences, split-ubiquitin, other domains used for protein interaction assays (described above), biotinylatio ⁇ sequence or other antibody epitope tags and protein purification domains such as His tags or GST.
  • the library may be used to screen for disruption or alteration of a specific biological process or cell phenotype.
  • positives may be selected based on detecting the disruption of the biological process (as an example, ability to grow on a specific substrate or medium) or cell phenotype.
  • interaction of the library member with the target may prevent the target from interacting with another cellular component or may prevent interactions between cellular components other than the target.
  • the library may be used to identify candidates that inhibit protein-protein interactions.
  • I N and l c refer to the N- and C-intein domains that flank the insert.
  • the modifications made to the int ⁇ in domains so that the inteins form a lariat, unprocessed intein, or dicysteine intein are discussed below.
  • the insert includes an insertion site for insertion of nucleic acid molecules encoding random peptides, ScFvs, or genomic fragments, as discussed below.
  • the insertion site may be for example a single restriction site, two adjacent restriction sites or a multiple cloning site as known in the art.
  • the insert may comprise an Nrul restriction enzyme recognition site although as will be apparent to one of skill in the art, any suitable restriction enzyme recognition site may be used. It is further noted that 'suitability' will be readily understood to one of skill in the art to include factors such as but by no means limited to uniqueness within the vector sequence and enzymatic activity.
  • PCR can also be used to generate a linearized lariat, unprocessed, or dicysteine vector for inserting nucleic acid molecules encoding random peptides, ScFvs, or genomic fragments, as discussed below.
  • a modified intein lariat library comprising a host- operable promoter operably linked to a nucleic acid molecule comprising, in order, an activity domain, a modified C-intein, an insert having a random peptide, ScFv, or genomic fragment encoding oligonucleotide inserted therein, a modified N-intein and a transcription termination sequence.
  • an oligonucleotide encoding one or more amino acids has been inserted into the insertion site of the insert.
  • the amino acid(s) encoded by the random peptide, ScFv, or genomic fragment encoding oligonucleotide will form the loop of the lariat, unprocessed intein or dicysteine intein.
  • random amino acid libraries do not need to contain all twenty amino acids. Libraries can consists on any combinations of two or more amino acids.
  • the modified intein libraries may be arranged for transformation into a suitable host or may comprise a mixture of host cells already transformed with the library as discussed below.
  • the modified intein lariat libraries are transformed into a suitable host or host cells or cell line.
  • the cells may be cells that have been previously transformed or transfected with a nucleic acid molecule encoding the target molecule fused to the second activity domain as discussed above.
  • the library may be introduced first and the target may be introduced second or the host may be co-transformed with the library and the target.
  • suitable hosts include but are by no means limited to bacteria, yeast, phage, Drosophilia Melanogaster, C. elegans, zebra fish, mice or other model organisms and mammalian cell lines, insect cell lines and the like.
  • a detectable product is produced and the specific intein library member can be recovered from the host cell expressing the detectable product and seque ⁇ ced.
  • a detectable product is produced and the specific intein library member can be recovered from the host cell expressing the detectable product and seque ⁇ ced.
  • any molecule that the activity domain can be attached to may be used as a target. It is of note that a large number of protein-protein interactions for a wide variety of peptides have been identified using the yeast two-hybrid system on which this method is based as discussed herein. As discussed herein, the intein sequences are modified to produce either a lariat structure, which undergoes a partial intein reaction, producing a lariat with a cyclical 'loop' and a N- terminal tail to which the activity domain is attached.
  • the unprocessed intein and dicysteine intein do not undergo any steps in the intein reaction and therefore the activity domain can be added to either the C-terminal of N-terminal end.
  • the lariat, unprocessed intein, and dicysteine intein are generated by making specific mutations to the intein sequences thereby blocking complete processing of the intein.
  • the I N intein domain is numbered (I N+1 , I N+2 , I N+3 ... ⁇ from N- terminus to C-terminus;
  • the extein is numbered from the C-terminus to the N-terminus ⁇ IN-I, IN-2, IN-3 ••• ⁇ or from the N-terminus to the C-terminus ⁇ lc + i, lc+2, lc+3 - ⁇ ; ⁇ »)
  • the l c intein domain is then ⁇ l C -i, lc-2, lc-3 ⁇ • ⁇ from the C-terminus to the N-terminus of the intein (Perler, F. B. (2002). Nucl. Acids Res. 30:383-384).
  • Blocks N1 , N3, EN1, EN2, EN3, EN4, C2 and C1 (Pietrokovski, S. (1998) Protein Sci. 7:64-71).
  • the present disclosure uses the A, B, C, D, E, H, F, G nomenclature and assigns each amino acid position in a conserved block a number from N-terminus to C-terminus. For example, l c+t , which is the eighth amino acid from the N-terminus in block G, is labelled G8. Similarly, I N+1, which is the first amino acid from the N-terminus of block A, is labelled A1.
  • the I N intein domain contains blocks A and B and the Ic intein domain contains blocks F and G.
  • the region to be cyclized or the extein is numbered from N-terminus to C-terminus ⁇ l c+ i, lc+2, lc + 3, ⁇ •, IN- 3 , IN-2, IN-I ⁇ (See Fig. 8 for overview of numbering scheme).
  • amino acids within 5 amino acids of the splice junctions will be named using both conventions i.e. l c+1 (G8).
  • Amino acids further than 5 amino acids from the splice site will be referred to by their conserved block and amino acid number.
  • the acyl shift introduces a thioester or an ester into the amide backbone of the peptide. E ⁇ ster bonds are more labile than amide bonds and thus provide a good leaving group for the reaction in Step 2 (Transesterification reaction).
  • Step 2 Transesterification reaction
  • either Cys, Ser, or Thr at position lc + i can act as a nucleophile that reacts with the thioester or ester bond formed in the Step 1 (N-X acyl shift).
  • Step 3 Asn cyclizatio ⁇ cleaves the amide bond that connects amino acids at positions l c+ i (G8) and l C -i (G7) and releases the extein, which contains a thioester or ester bond between I 0+1 (G8) and l N .i).
  • GIn also occurs at position l c .i (G7) and undergoes a similar cyclization reaction (Pietrokovski, S.
  • Step 1 (N-X acyl shift) is not needed in inteins that use the non-standard mechanism since the amide bond is already aligned for direct attack by the nucleophile at Ion (G8) and therefore they do not need the extension in the backbone caused by Step 1 (N-X acyl shift) (Southworth, M.W. et al. (2000) EMBO J. 19:5019-5026, Tru, B.W. et al. (2000) J. Biol. Chem. 275:16408-16413).
  • inteins may also use a mechanism lor intein splicing that is different from the standard mechanism. For example, Asp has been identified at loi (G7) in place of Asn (1/344) (Amitai, G. et al. (2004) J. Biol. Chem. 279:3121-3131). This intein may undergo Asp cyclization at Step 3 (Asn cyclization) of the standard mechanism.
  • inteins that contain other amino acids at indicated sites include: GIn (2/344) (Cf ⁇ TerA and PhiEL ORF11 inteins), Met (1/344) (PNEL ORF40 intein), and Pro (1/344) (Mbe DnaB intein) at position I N+1 (A1); VaI (2/344) (Cth ATpase and Pfi Fha), GIy (1/344) (Avin P.IR1), and Tyr (1/344) (Mmag Magn8951) at position ⁇ M (G8); and His (1/344) (Mga SufB (Mga Pps1 )) at position lc-i (G7).
  • mutant inteins described in this invention refer to mutants in the intein-mediated protein cyclization reaction (Fig. 11). These mutants are referred to as the unprocessed intein, the dicysteine intein, and the lariat intein. These three mutant inteins are described by the mutations required to generate them.
  • Step 2 Transesterification reaction
  • the transesterification reaction releases the I N domain. If the I N domain is released, then the I 0 - I N domain interaction responsible for the scaffolding ability of this mutant is disrupted.
  • Step 1 N-X acyl shift
  • Step 1 N-X acyl shift
  • Step 1 results in the formation of a thioester or ester bond at the Extein-I N junction, between I N-1 and IN +I (A1 ).
  • the thioester or ester bond is more susceptible to hydrolysis than an amide bond.
  • Dicysteine int ⁇ ins have Cys at positions loi (G8) and l N+) (A1) that are used to cross link peptides, genomic fragments, or ScFvs that interact with a target. Since these amino acids can function as nucleophiles in Step 1 (N-X acyl shift) and Step 2 (Transesterification reaction) of the intein reaction, strategies are needed that inhibit these steps without mutating these Cys. At minimum, Step 2 (Transesterification reaction) needs to be inhibited to prevent formation of an unstable thioester bond between l c+ i (G8) and I N - ! the last residue of the extein, which results in the cleavage of the I N domain.
  • the dicysteine intein can be stabilized by inhibiting Step 1 (N-X acyl shift), which prevents the hydrolysis of the Extein-I N ester or thioester.
  • the dicysteine intein can be further stabilized by inhibiting Step 3 (Asn cyclization), which prevents Ic- Extein cleavage between Ic -1 (G7) and l c+ i (G8).
  • the lariat Intein is generated by inhibiting the Step 3 (Asn cyclization) in the intein reaction.
  • the lariat intein is cyclized through a lactone bond, which is more susceptible to hydrolysis than an amide bond.
  • the lariat can be further stabilized by inhibiting hydrolysis of the lactone bond.
  • the strategies can be used either alone or in combinations to generate unprocessed inteins, dicysteine inteins, or lariat inteins.
  • Step 1 N-X Acyl Shift
  • the N-X acyl shift involves the l N+ i (A1) nucleophile, which is usually Ser or Cys. Although Thr is not normally present at position I N+ , (A1), it could also potentially function as a nucleophile in Step 1 (N-X acyl shift).
  • Step 1 N-X acyl shift
  • Step 1 produces an ester or thioester bond that replaces the amide bond between the I N+1 (A1) residue and the l N -i residue (the last residue of the extein).
  • the ester or thioester forms a good leaving group for Step 2 (Transesterification), however the ester or thioester bond is susceptible to hydrolysis, which can result in cleavage between the Extein-l N at I N _, and I N+1 (A1). Therefore, if Step 2 (Transesterification) is inhibited, I N cleavage by hydrolysis can become a side product. Mutation of amino acids that are involved in catalyzing the N-X acyl shift can block Step 1 in the intein reaction.
  • the catalytic pocket where the N-X acyl bond is formed contains amino acids in Block B: B7 (Thr ⁇ 5 ⁇ 0 " ⁇ , ThtfO 5 *" 0 TM 6 ), B9 (Asn72 &p DrtaB ), B10 (His72 & " OnaE , HisT ⁇ *" DnaB ), amino acids in Block F: F2 (VaH 34 s * Dri ⁇ B ), F3 (Phe139 Ssp D ⁇ aE ), F4 (Asp140 s *" 0 ⁇ ), amino acids between Blocks A and B: ArgSO ⁇ DnaE , ThrSi ⁇ 0 ⁇ , Lys54 Sv DnaB , the nucleophile in Block A: A1 ⁇ Cysi Ssp DnaE ), the adjacent amino acid in Block A: A2 (Leu2 Ssp 0 ⁇ ), and the last residue of the extein: I N ., (Sun, P.
  • Step 1 N-X acyl shift
  • These strategies can be used to generate the unprocessed intein and the dicysteine intein.
  • Strategy 1.2 Mutation of the F3 amino acid. Analysis of Ssp DnaE, Pl Seel, and Ssp DnaB intein structures reveals that amino acids at position F3 are in the catalytic pocket where the N-X acyl bond is formed. Mutation of Phe at position F3 in Ssp DnaE to Ala inhibits the formation of the ester or thioester between I N-1 and l N+ i (Ghosh, I. et al. (2001) J. Biol. Chem. 276:24051-24058).
  • mutations of of amino acids within hydrogen bonding distance of Wi may be used to disrupt Step 1 (N-X acyl shift) or Step 2 (Transesterification reaction).
  • mutations that block Step 1 may accordingly include substitutions at positions B9, B10, or F2 (the equivalent amino acids to Arg50 SspDnaE and Thr51 in SspDnaB), including substitution of non-catalytic amino acids at these positions.
  • the transesterification reaction involves nucleophilic amino acids at position Ic +1 (G8) attacking the ester or thioester bond formed in Step 1 (N-X acyl shift), which results in the formation of a ester or thioester bond between l c+ i (G8) and 1 ⁇ 1 .
  • the transesterification reaction releases the I N domain from the l c -extein domain (Split intein product).
  • the ester or thioester bond formed between the l c+ i (G8) residue and l N -i can potentially be hydrolysed resulting in a linear intein product consisting of l c -extein (Split intein product).
  • Ic t2 and I N -I are found in the catalytic pocket for the transesterification reaction and can potentially influence splicing.
  • Step 2 Transesterification reaction
  • Step 2 Transesterification reaction
  • Ser I 0+ I G8
  • Cys or Thr Xu, M. & Perler, F.B. (1996) EMBO J. 15:5146-5153
  • mutation of Cys at position l c+ i (G8) to Ser inhibits Step 2 (Transesterification reaction) in the See VMA1 intein (Hirata, R. & Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 188:40-47).
  • Strategy 2.4 Introduction of a charged amino acid near the splice sites. Mutation of Leu at position I N+2 (A2) in Psp Pol or mutation of Ala at position l C - 2 (G6) in Psp Pol to Lys prevents cleavage of the I N domain (Xu, M. & Perler, F.B. (1996) EMBO J. 15:5146-5153). Mutation of VaI at position I 0-2 (G6) to Arg or Phe blocks splicing in the See VMA intein, however, Ser, Cys, lie, and GIy mutations do not inhibit splicing (Cooper, A.A. et al. (1993) EMBO J. 12:2575-2583). Mutations that introduce a charge at positions I N+2 (A2) and lc- 2 (G6) should inhibit the transesterification reaction.
  • Zinc-mediated inhibition Zinc coordinates with the Ic +1 (G8) nucleophile and prevents splicing (Mills, K. V. & Paulus, H. (2001) J. Biol. Chem. 276:10832-10838). Addition of Zinc at concentrations greater than 10 ⁇ M should block the transesterification reaction.
  • Strategy 2.7 The amino acid at position F6 coordinates the Ser (G8) for attack on the thioester formed in Step"! . Accordingly, mutation at position F6 to a non- catalytic residue may be used to block Step 2.
  • Step 3 Asn Cyclization: Step 3 (Asn cyclization) results in cleavage of the l c domain from the extein.
  • the most common mechanism for this step involves Asn cyclization. This mechanism is used by 327 of the 344 inteins in the InBase database.
  • the second most common method involves GIn cyclization, which is used by 15 of the 344 inteins in the InBase database.
  • Block B B11 (Arg73 s * D ⁇ aE ); Block F: F5 (Leu137 s * 0 ⁇ ), F6 (Thr138 S5P DnaB ), F7 (VaUS ⁇ 0 ⁇ 8 , Leu143 5 ⁇ 00 ⁇ ) 1 F13 (His143 Ssp D ⁇ aB , His147 Ssp OnaE ); Block G: G6 (His153 s * 0 ⁇ 8 ), G7 (Asn154 Sip DnaB , Asn159 ⁇ DnaE ), G8 (Ser155 Ssp °" aE , Cys160 Sjp DnaE ), the second residue of the extein (Ic +2 ), and the last residue of the extein (I N-1 ) (Sun, P. et al. (2005) J. MoI. Biol. 353:1093-1105, Ding,
  • Step 3 Mutation of Asn at position l c -i (G7) in the See Tfp1 intein to Lys, Ala, Tyr, GIn, GIu, His, and Asp all inhibit Step 3 (Asn cyclization) (Cooper, A.A. et al. (1993) EMBO J. 12:2575-2583).
  • mutation of Asn at position I ⁇ -i (G7) to hydrophobic amino acids may also stabilize the ester or thioester formed in Step 2 (Transesterification reaction). This prediction is based on the observed accumulation of branched intermediate when His at position l c .
  • G6 is mutated to a Leu, Asn, or GIn (Xu, M. & Perler, F.B. (1996) EMBO J. 15:5146-5153). Therefore, certain mutations of Asn at position l c -i (G7) may stabilize the lariat.
  • G6 binds to the Asn carbonyl oxygen at position l c- i (G7).
  • Arg at position B11 binds to the Asn carbonyl oxygen at position l C -i (G7) (Ding, Y. et al. (2003) J. Biol. Chem. 278:39133-39142).
  • the use of His or Arg to interact with the Asn carbonyl oxygen depends on residues in the extein.
  • Phe at position (IC + ⁇ ) and Phe at position (I N ⁇ ) in the extein form a hydrophobic pocket that interacts with the imidazole ring of His at position l C - 2 (G6), which prevents it from interacting with the Asn carbonyl oxygen at position Ic-, (G7) (Sun, P. et al. (2005) J. MoI. Biol. 353:1093-1105). Mutation of His at position lc. 2 (G6) in the Psp pol-l intein to Leu, Asn, and GIn results in an accumulation of the branched intermediate (Xu 1 M. & Perler, F.B. (1996) EMBO J. 15:5146-5153).
  • Strategy 3.3 Mutation of the amino acids at position F13. Mutation of His at position F13 in the Ssp DnaB intein to GIn blocks Step 3 (Asn cyclization) (Ding, Y. et al. (2003) J. Biol. Chem. 278:39133-39142). Mutation of His at position F13 in the Ssp DnaB intein to Ala only partially inhibits Step 3 (Asn cyclization) (Ding, Y. et al. (2003) J. Biol. Chem. 278:39133-39142).
  • Strategy 3.5 Mutation of the amino acids at position F15.
  • the amino acid at position F15 is highly conserved.
  • Mutation of Phe at position F15 in the Ssp DnaB intein to Ala blocks Step 3 (Asn cyclization) (Ding, Y. et al. (2003) J. Biol. Chem. 278:39133-39142).
  • Mutation of Phe at position F15 in the Ssp DnaB intein to Tyr slightly inhibits Step 3 (Asn cyclization) (Ding, Y. et al. (2003) J. Biol. Chem. 278:39133-39142).
  • a mixed intein containing the I N domain from Npu DnaE intein and an l c domain from Ssp DnaE intein is much more tolerant to amino acid substitutions at this position (Iwai, H. et al. (2006) FEBS Lett. 580:1853-1858). Therefore fixing this amino acid in random libraries may be beneficial when using certain inteins.
  • Amino acids at position I N-1 are found in the N-X acyl shift catalytic pocket (Sun, P. et al. (2005) J. MoI. Biol. 353:1093-1105). In Ssp DnaE, Tyr at position I N . !
  • Step 3 (Asn cyclization) from occurring before Step 2 (Transesterification reaction) is finished
  • a modified See VMA intein used for protein purification is fused C-terminal to the target protein, the See VMA is mutated to prevent Step 3 (Asn Cyclization) and Step 2 (Transesterification reaction) allowing only Step 1 (N-X acyl shift) to occur.
  • Certain amino acids at position I N ., in See VMA intein allow Step 1 (N-X acyl shift) to occur in vivo: Thr, GIu 1 His, Arg, and Asp.
  • Step 1 N-X acyl shift
  • Step 3 Asn cyclization
  • Step 3 Asn cyclization
  • Step 2 Transesterification
  • Step 2 may be blocked by mutating Ser at position l c+ i (G 8) to Ala. It may also be blocked by mutating Ser at position I 0+I (G8) to other amino acids.
  • a strategy refers to a mutation, there may be multiple amino acid substitutions at that site that will accomplish the same outcome.
  • the application of the strategies 2.1 - 2.6 defined above to the unprocessed intein are described below.
  • the amino acid at position l c+ i needs to be mutated to an amino acid that cannot function as a nucleophile in Step 2 (Transesterification reaction).
  • the inteins listed in InBase contain Ser, Cys, and Thr at position l c+ i ⁇ G8). Mutation of ⁇ M (G8) to any other amino acid should inhibit Step 2 (Transesterification reaction).
  • certain inteins are only able to use a specific nucleophilic amino acid at position I 0+1 (G8) (Shingledecker, K. et al. (2000) Archives Biochem. Biophys. 375:138-144).
  • Step 2 Transesterification reaction
  • Step 2 Transesterification reaction
  • Psp PoI-I intein Step 2 (Transesterification reaction) is inhibited, when Ser I 0+I (G8) is mutated to Cys or Thr, (Xu, M. & Perler, F. B. (1996) EMBO J. 15:5146-5153).
  • the amino acid at position B7 needs to be mutated to an amino acid that cannot hydrogen bond to the carbonyl oxygen at position I N+1 (A1).
  • the following amino acids at position B7 occur more than once in the Inteins listed in InBase: Thr, Ser, Asn, Asp, Cys, and GIu. Mutation of the amino acids at position B7 to any other amino acid except Thr, Ser, Asn, Asp, Cys, and GIu, should inhibit Step 2 (Transesterification reaction).
  • certain inteins are able to only use a specific amino acid at position B7. Therefore, for these inteins, Step 2 (Transesterification reaction) can be inhibited by substituting the wild type amino acid for any other amino acid at position B7.
  • the amino acid at position B10 needs to be mutated to an amino acid that cannot hydrogen bond with the amido nitrogen at position l N+ i (A1).
  • the most common amino acids at position B10 in inteins listed in InBase that are believed to undergo splicing are His and Thr.
  • the amino acids Asp and Lys also occur at position B10 although at a much lower frequency than His and Thr.
  • These amino acids are capable of hydrogen bonding with the amido nitrogen at position I N+1 (A1) and mutation of amino acids at position B10 to any other amino acid except His, Thr, Asp, and Lys should inhibit Step 2 (Transesterification reaction).
  • certain inteins are able to only use a specific amino acid at position B10. Therefore, for these inteins, Step 2 (Transesterification reaction) can be inhibited by substituting the wild-type amino acid for another amino acid at position B10.
  • a charged amino acid is introduced near the splice site.
  • the inteins listed in InBase that are believed to undergo splicing contain primarily Leu, VaI, Phe, and He at position A2 (l N+ 2)-
  • the amino acids His, GIn, Met, GIy, Cys, Ser, Thr, and Tyr occur in less than ten inteins at position A2 (I N+2 ).
  • the most frequently occurring amino acids are VaI, Thr, Leu, Ala, and Ser.
  • the amino acids Cys, lie, Asn, and His occur in four or less inteins.
  • Step 2 Transesterfication reaction
  • inteins are able to only use a specific amino acid at positions A2 (I N+2 ) and G5 (l C -a). Therefore, for these inteins, Step 2 (Transesterification reaction) can be inhibited by substituting the wild- type amino acid for another amino acid at positions A2 (I N+2 ) and G5 (Ic ⁇ ).
  • the amino acid at position F4 needs to be mutated to an amino acid that cannot hydrogen bond with the carbonyl oxygen of the I N-1 .
  • the inteins listed in inBase that are believed to undergo splicing primarily contain Asp, Cys, Thr, Trp, Ser, and Asn at the F4 position.
  • Amino acids Arg, Ala, GIu, Phe, GIy, Me, Leu, GIn, VaI, and Tyr occur in five or less inteins.
  • the amino acids Asp, Cys, Thr, Trp, Ser, and Asn can all form hydrogen bonds with the carbonyl oxygen of the I N-1 .
  • Step 2 Transesterification reaction
  • certain inteins are able to only use a specific amino acid at position F4. Therefore, tor these inteins Step 2 (Transesterification reaction) can be inhibited by substituting the wild-type amino acid for another amino acid at position F4.
  • the loi (G8) nucleophile needs to be Cys.
  • the l c+ i (G8) nucleophile is Cys, addition of Zinc to the growth media will inhibit Step 2 (Transesterification reaction).
  • Step 1 N-X acyl shift.
  • strategy 1.1 - 1.3 for generating unprocessed intein are described below.
  • the amino acid at position A1 (I N+1 ) needs to be mutated to an amino acid that cannot function as a nucleophile in Step 1 (N-X acyl shift).
  • the inteins listed in In Base that are believed to undergo splicing primarily contain Cys, Ser, and to a lesser extent Ala. Inteins with Ala at this position undergo the alternative intein mechanism described above. In standard inteins, mutation of the amino acid at position A1 to any other amino acid should inhibit Step 1 (N-X acyl shift).
  • Step 1 N-X acyl shift
  • the Psp PoI-I intein splices poorly when Ser I N+1 (A1) is mutated to Cys, and splicing is blocked completely when Ser is mutated to Thr (Xu, M. & Perler, F.B. (1996) EMBO J. 15:5146-5153).
  • the amino acid at position F3 needs to be mutated to an amino acid the disrupts N-X catalytic pocket.
  • the inteins listed in InBase that are believed to undergo splicing primarily contain Tyr and Phe at the F3 position.
  • Amino acids GIu, lie, Arg, VaI, GIn, Asp, Lys, Thr, His, Leu, Trp, Ser, Cys, GIy, Asn, and Pro occur less often at this position. Mutation of amino acids at position F3 to any amino acid other than Tyr or Phe will inhibit Step 1 (N-X acyl shift).
  • certain inteins are able to only use a specific amino acid at position F3. Therefore, for these inteins, Step 1 (N-X acyl shift), can be inhibited by substituting the wild-type amino acid for another amino acid at position F3.
  • Step 1 amino acids within hydrogen bonding distance of the side chain of the I N+1 (A1) nucleophile need to be mutated.
  • the amino acids found here do not correspond to amino acids in the conserved intein blocks.
  • Thr and Arg are within hydrogen bonding distance of the side chain of the l N+ i (A1) nucleophile in the Ssp DnaE and Ssp DnaB inteins. Mutation of Thr or Arg to an amino acid that cannot hydrogen bond to the side chain of the I N+1 (A1) nucleophile will inhibit Step 1 (N-X acyl shift), or Step 2 (Transesterification).
  • Step 1 N-X acyl shift
  • Step 3 Asn Cyclization
  • a stable unprocessed intein can be generated using a single or a combination of strategies that inhibit Step 1 (N-X acyl shift) with a single or combination of strategies that inhibit Step 3 (Asn Cyclization).
  • There are three strategies (1.1 - 1.3) to inhibit Step 1 (N-X acyl shift) and five strategies (3.1 - 3.5) to inhibit Step 3 (Asn cyclization), which results in a total of (ZM) x (2 5 -1) 217 different strategies for inhibiting Stops 1 (N-X acyl shift) and 3 (Asn Cyclization).
  • Application of the strategies 1.1 - 1.3 for generating unprocessed intein are described above.
  • Application of strategies 3.1 - 3.5 for generating unprocessed intein are described below.
  • the amino acid at position I 0 - I (G7) needs to be mutated to an amino acid that cannot undergo cyclization.
  • the inteins listed in InBase that are believed to undergo splicing contain Asn and GIn at position I 0 - I (G7). Mutation of amino acids at position l C -i (G7) to an amino acid that cannot undergo side chain cyclization will inhibit Step 3 (Asn cyclization). However, certain inteins are able to only use a specific amino acid at position l C -i (G7). Therefore, for these inteins, Step 3 (Asn cyclization) can be inhibited by substituting the wild-type amino acid for another amino acid at position lc-i (G7).
  • amino acids G6 (l C - 2 ) and/or B11 which assist in Asn cyclization by hydrogen bonding with the Asn carbonyl oxygen at position l c .i (G7) should be mutated to an amino acid that cannot hydrogen bond with this amino acid.
  • the inteins listed in InBase that are believed to undergo splicing contain His at the G6 (Ic-.) position and to a lesser extent GIy, Ser, Ala, and Cys. Mutation to any amino acid except for His should inhibit Step 3 (Asn cyclization).
  • B11 can assist in Asn cyclization by hydrogen bonding with the Asn carbonyl oxygen at position l C -i (G7).
  • B11 is predominately Lys or Arg when G6 is not His. Mutation to any amino acid that does not have a positive charge (Lys, Arg, or His) at either position should inhibit Step 3 (Asn cyclization)
  • certain inteins are able to only use a specific amino acid at position G6 (I 0 *) or B11. Therefore, for these inteins, Step 3 (Asn cyclization) can be inhibited by substituting the wild-type amino acid for another amino acid at position G6 (lc.2) or B11.
  • the amino acid at position F13 needs to be mutated to an amino acid that cannot act as a proton acceptor from Asn at position l C -i
  • Step 7 through a coordinated water molecule.
  • the inteins listed in InBas ⁇ that are believed to undergo splicing contain primarily His, and to a lesser extent, GIu, GIn, Asn, Pro, Ser, Lys, AIa 1 GIy, Asp, Arg, Me, Leu, Tyr, Trp, VaI, and Thr at position F13. If the wild-type residue is Mis mutation to another amino acid should inhibit Step 3 (Asn cyclization). However, certain inteins are able to only use a specific amino acid at position F13. Therefore, for these inteins, Step 3 (Asn cyclization) can be inhibited by substituting the wild-type amino acid for another amino acid at position F13.
  • the amino acid at position F14 needs to be mutated to an amino acid that inhibits Asn cyclization.
  • the inteins listed in In Base that are believed to undergo splicing contain primarily Asn at position F14.
  • the amino acids, Leu, Ser, Thr, GIn, Ala, Arg, Met, Phe, VaI, GIu, Tyr, His, Lys, Cys, Asp, and Me occur less frequently. Mutation to any other amino acid would disrupt the splice site inhibiting Step 3 (Asn cyclization).
  • certain inteins are able to only use a specific amino acid at position F14. Therefore, for these inteins, Step 3 (Asn cyclization) can be inhibited by substituting the wild type amino acid for another amino acid at position F14.
  • the amino acid at position F15 needs to be mutated to an amino acid that inhibits Asn cyclization.
  • the inteins listed in InBase that are believed to undergo splicing contain primarily Phe and Tyr at position F15.
  • the amino acids VaI, GIy, Asn, Ser, Thr, His, lie, Trp, Ala, and GIu also occur at position F15.
  • the F15 position forms hydrophobic contacts with amino acids surrounding the splice site and orients the amino acid at position F13. Mutation of the amino acid at position F15 from Phe or Tyr to any amino acid except Phe or Tyr will inhibit Step 3 (Asn cyclization).
  • Step 3 (Asn cyclization) can be inhibited by substituting the wild type amino acid for another amino acid at position F15.
  • Step 1 N-X acyl shift
  • Step 2 Transesterification
  • Three strategies (1.1 - 1.3) can be used to inhibit Step 1 (N-X acyl shift)
  • l c+ i G8
  • Step 2 Transesterlflcation
  • Step 3 Analog of Step 3
  • Step 1 N-X acyl shift
  • Step 2 Transesterification
  • Step 3 Asn Cyclization
  • the application of the strategies 1.1 - 1.3, 2.1 - 2.6, and 3.1 - 3.5 for the unprocessed intein are described above.
  • the dicysteine intein does not undergo any steps in the intein-mediated splicing reaction (Fig. 13). Cys amino acids at positions l c+ i (G8) and IN +1 (A1) are retained and other mutations are required to inhibit intein processing. After dicysteine inteins are selected that interact with a given target, a peptide containing the random peptide, ScFv, or genomic fragment flanked by the cysteine residues can be synthesized. These peptides, ScFvs, and genomic fragments can then be constrained by disulfide bonds or cysteine cross- linking reagents.
  • the Cys amino acids at positions I 0+ I (G8) and l N+ i (Ai) are required for the dicysteine intein.
  • Strategy 2.2 Zinc inhibition is a good strategy for generating the dicysteine intein as it does not require mutation at lc + i (GB) 1 and inhibition is reversible.
  • an intein that is not tolerant to substitutions at l c+ i (G8) and I N+1 (A1) can be used to generate the dicysteine intein.
  • the Psp Pol intein has Ser at positions lc + i and l N+ i and mutation to Cys inhibits protein splicing (Xu, M. & Perler, F. B. (1996) EMBO J. 15:5146-5153).
  • Dicysteine intein can be generated using a single strategy or a combination of strategies that inhibit only Step 1 (N-X acyl shift).
  • There are three strategies (1.1 - 1.3) to inhibit Step 1 (N-X acyl shift), which gives rise to 2 3 - 1 7 strategies for inhibiting Step 1 (N-X acyl shift).
  • the application of the strategies 1.1 - 1.3 for the dicysteine intein are described below.
  • Step 1 N-X acyl shift
  • Psp PoI-I intein splices poorly when Ser I N+1 (A1) is mutated to Cys (Xu, M. & Perler, F.B. (1996) EMBO J. 15:5146-5153).
  • the amino acid at position F3 needs to be mutated to an amino acid the disrupts N-X acyl shift catalytic pocket.
  • Inteins listed in InBase that are believed to undergo splicing primarily contain Tyr and Phe at the F3 position.
  • Amino acids GIu, lie, Arg, VaI 1 GIn, Asp, Lys, Thr, His, Leu, Trp, Ser, Cys, GIy, Asn, and Pro occur less often at this position. Mutation of amino acids at position F3 to any amino acid other than Tyr or Phe should inhibit Step 1 (N-X acyl shift).
  • Step 1 N-X acyl shift
  • Step 1 N-X acyl shift
  • Step 3 Asn Cyclization
  • a stable dicysteine intein can be generated by using a single or a combination of strategies that inhibit Step 1 (N-X acyl shift) with a single or combination of strategies that inhibit Step 3 (Asn Cyclization).
  • There are three strategies to inhibit Step 1 (N-X acyl shift) (1.1 - 1.3) and five strategies to inhibit Step 3 (Asn Cyclization), which results in a total oi (2 3 -1) x (2 s - 1) 217 strategies to inhibit Steps 1 (N-X acyl shift) and 3 (Asn Cyclization). If the intein has a Cys at position l N+ i (A1), then Strategy 1.1 is not relevant.
  • a stable dicysteine intein can be generated by using a single or a combination of strategies that inhibit Step 1 (N-X acyl shift) with a single or combination of strategies that inhibit Step 2 (Transesterification).
  • Step 1 N-X acyl shift
  • Step 2 Transesterification
  • There are three strategies to inhibit Step 1 (N-X acyl shift) (1.1 - 1.3) and six strategies to inhibit Step 2 (Transesterification), which results in a total of (2M) (2 ⁇ -1) 441 strategies to inhibit Steps 1 (N-X acyl shift) and 2
  • Step 2 Transesterification
  • Step 2 Transesterification reaction
  • a stable dicysteine intein can be generated by using a single or a combination of strategies that inhibit Step 2 (Transesterification) with a single or combination of strategies that inhibit Step 3 (Asn cyclization).
  • There are six strategies (2.1 - 2.6) to inhibit Step 2 (Transesterification) and five strategies (3.1 - 3.5) to inhibit Step 3 (Transesterification), which results in a total of (2 6 -1) x (2 5 -1) 1953 strategies to inhibit Steps 2 (Transesterification) and 3 (Asn cyclization).
  • strategy 2.1 is not relevant, which leaves five strategies (2.2 - 2.6) to inhibit Step 2 (Transesterification).
  • Step 1 N-X acyl shift
  • Step 2 Trans ⁇ st ⁇ rfication
  • Step 3 Asn cyclization
  • a stable dicysteine intein can be generated by using a single or a combination of strategies that inhibit Step 1 (N-X acyl shift), with a single or combination of strategies that inhibit Step 2 (Transesterification reaction), and with a single or combination of strategies that inhibit Step 3 (Asn cyclization).
  • the lariat intein is generated by allowing the first two steps of intein reaction (Fig. 14) to proceed and by blocking the third step, (Asn cyclization). Any residues surrounding I 0+1 (G8) that stabilizes the ester bond from hydrolysis should also be incorporated. Mutations that enhance the first two steps are also beneficial. Strategy 4 may also be beneficial for generating robust lariat libraries, where an increased number of library members form lariats.
  • GIn Asp
  • Asp Asp
  • G6 amino acids at position G6 (I 0 2 ) are mutated to Leu, Asn, or GIn (Xu, M. & Perler, FB. (1996) EMBO J. 15:5146-5153).
  • G6 (l C - 2 ) and/or B11 which assist in Asn cyclization by hydrogen bonding with the Asn carbonyl oxygen at position l C -i (G7) should be mutated to an amino acid that cannot hydrogen bond with this amino acid.
  • the inteins listed in InBase that are believed to undergo splicing contain His at the G6 (lc- 2 ) position and to a lesser extent GIy, Ser, Ala, and Cys. Mutation to any amino acid except for His should inhibit Step 3 (Asn cyclization). In the absence of the His at G6 (lc. 2 ), it has been found that B11 can hydrogen bonding with the Asn carbonyl oxygen at position l C -i (G7).
  • Position B11 is predominately Lys or Arg when G6 is not His. Mutation to any amino acid that does not have a positive charge (Lys, Arg, His) at either position should inhibit Step 3 (Asn cyclization). Mutation of His at position Ic- 2 (G6) in the Psp pol-l intein to Leu, Asn, and GIn results in an accumulation of the branched intermediate only when lc-i (G7) is not mutated to Ala (Xu 1 M. & Perler, RB. (1996) EMBO J. 15:5146-5153). Currently, there are no mutagenic studies on the role of Arg at position B11 in accumulating branched intermediates.
  • Step 3 (Asn cyclization) can be inhibited by substituting the wild-type amino acid for another amino acid at positions G6 (lc. 2 ) or B11.
  • the present invention describes the construction and application of the "lariat", unprocessed intein, and dicysteine intein in the yeast two-hybrid assay.
  • the lariat is a new peptide construct that has no C-terminus and represents a novel class of cyclic peptides. Lariat peptides are generated by modifying the in vivo intein-mediated protein ligation reaction. The C-terminus of the lariat peptide is looped back and linked to a specific serine in the interior of the peptide via a cyclic lactone bond (Fig. 3). The lariat has a free N- terminus that allows the attachment of useful biological domains such as an activation domain, which is necessary for yeast-two hybrid assays.
  • LexA represents a putative antimicrobial target, which when inhibited should potentiate that activity of cytotoxic antibiotics.
  • LexA is bound by activated RecA it undergoes autoprot ⁇ olysis and no longer represses genes in its regulon (Lin, LL. & Little, J.W. (1988) Bacterid. 170:2163-2173).
  • LexA mutants that block autoproteolysis (Walker, G.C. (1984) Microbiol.
  • Lariats were generated that are compatible with the yeast two-hybrid system by engineering the intein producing cyclic peptide system (Scott, CP. et al. (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643) to halt the cyclic peptide reaction at an intermediate step, which produces a lariat that contains a transcription activation domain covalently attached through an amide bond to a lactone-cyclized peptide.
  • a library of approximately seven million lariat peptides was constructed in the MATa yeast strain EY93 (FIg. 16) and mated the library to a MAT ⁇ strain EY111 containing the LexA target plasmid (pEG202) (Gyuris, J. et al. (1993) Cell 75:791 -803) and yeast two-hybrid reporter genes.
  • pEG202 LexA target plasmid
  • yeast two-hybrid interaction trap in Figure 15c, 14 clones were isolated, encoding two unique lariats that interacted with LexA (FIg. 15d).
  • the L2 lariat was used for further analysis as it contains more charged amino acids, which may enhance its solubility.
  • the noose region from the L2 lariat was cloned into an inactive lariat intein plasmid (plN-L2), which does not undergo any steps in the intein-mediated cyclization reaction.
  • plN-L2 inactive lariat intein plasmid
  • plL-L2 produces unprocessed (- 23 kDa) and lariat ( ⁇ 9 kDa) products
  • inactive intein plasm id plN-L2 produces only unprocessed product.
  • the lariat structure is important for the L2 lariat-LexA interaction, as activation of the yeast two-hybrid reporter genes with the inactive L2 intein (plN-L_2), expressing the unprocessed lariat, is barely detectable relative to the L2 lariat (pll_-L2), which expresses both the unprocessed product and the lariat (FIg. 17b).
  • MS mass spectrometry
  • MMC is a potent inducer of bacterial SOS response that activates the FtecA coprotease activity and induces cleavage of LexA (Lin, LL. & Little, J.W. (1988) Bacteriol. 170:2163-2173).
  • pETIL-L2 into BL21-CP and used Western analysis to monitor degradation of LexA after exposure to MMC in the presence and absence of the L2 lariat (Yasuda, T. et al. (1998) EMBO J. 17:3207-3216) (Fig. 21a).
  • LexA cleavage is not observed after three hours in cells that express L2 lariat, whereas in cells that express a lariat intein with a CPGC amino acid noose (pE ⁇ TIL-01) LexA is completely cleaved after one hour.
  • L2 lariat was expressed in BL21-CP cells, exposed the bacteria to MMC in 0.85% NaCI for one hour, and assayed their survival (Fig. 21c).
  • pETIL-L2 L2 lariat
  • pETIL-01 a lariat with a CPGC noose
  • Expression of either plasmid reduces the viability to - 35% of the uninduced controls.
  • MMC alone 0.1 ⁇ g/mL
  • Expression of L2 lariat enhanced the activity of MMC and reduced the viability to ⁇ 1% of the control, whereas expression of the lariat with CPGC noose did not enhance the activity of MMC.
  • the invention provides methods to genetically select lariats against a given target protein using intein-mediated peptide cyclization and the yeast two-hybrid interaction trap.
  • This system allows lariats and cyclic peptides based on the noose sequence of the lariats to be rapidly generated against protein targets that are compatible with the yeast two-hybrid system.
  • the lariat technology provides a rapid high throughput system for isolating cyclic peptide inhibitors that can be used for the reverse analysis of protein function or as drugs or pseudo-drugs for validating therapeutic targets.
  • lariat inhibitors of LexA We used this system to generate lariat inhibitors of LexA and validate LexA as a therapeutic target for potentiating the antimicrobial effects of reagents that activate the SOS response pathway.
  • the lariats can be converted to cyclic or linear peptides that also potentiate the effects of MMC.
  • Linear peptides are from the University of Calgary Rapid Multiple Peptide Synthesis Service (Calgary, AB). Cyclic peptides are from Anygen Co. Ltd. (Korea). Oligonucleotides are from IDT DNA (Coralville, IA) and are listed in Supplementary Table 1 online.
  • E.coli strains BL21(DE3) is from Novagen (Madison, Wl) and BL21-CodonPlus®(DE3)- RIL (BL21 -CP) is from Stratagene (La JoIIa, CA). SMR6039 is a gift from Susan Rosenberg (Hastings, PJ. et al. (2004) PLoS Biol. 2:e399).
  • EY93 MATa ura2 his3 trp1 Ieu2 ade2::URA3 is a derived from EGY42 (Cohen, B.A. et al. (1998) Proc. Natl. Acad. Sci. USA 95:14272-14277).
  • EY111 MAT ⁇ his3 trp1 ura3::LexA8op-lacZ ade2::URA3-LexA8op-ADE2 leu2::LexA6op-LEU2
  • EGY48 Golemis, E.A. & Brent, R. (1992) MoI. Cell. Biol. 12:3006-3014.
  • plN01 The lariat i ⁇ tein design is based on the amino acid sequence of the Synechocystis spp. strain PCC6803 (Ssp) DnaE intein gene. We assembled the inactive intein gene by mixing 0.1 ⁇ g of each of the eight oligonucleotides [A-H] with 2.5 units of pfu polymerase
  • KCI 0.1 % (v/v) Triton X-100, 0.1 mg/mL bovine serum albumin (BSA), and 2 mM MgSO 4 .
  • Lariat Library (plL-XX): We replaced the CPGC linker peptide in plN01 with a combinatorial seven amino acid peptide using oligonucleotide K. We PCR amplified oligonucleotide K using primers L and M. We used the reaction conditions described above with seven amplification cycles consisting of a denaturing step at 95 0 C for 30 seconds, an annealing step at 55 0 C for 30 seconds, and an extension step at 72 0 C for 15 seconds. We digested plN01 with Rsrll (New England Biolabs, Ipswich, MA) and dephosphorylated the digested plasmid with 10 units of shrimp alkaline phosphatase (Fermentas).
  • Rsrll New England Biolabs, Ipswich, MA
  • plL-L2 is a library member from the pi LXX library.
  • the noose sequence is (RSWDLPGEY).
  • plN-L2 We constructed plN-L2 by mutating cysteine at I N +1 to alanine, which produces an inactive intein. Two overlapping PCR fragments were used to introduce the point mutation. We used primers I and N to amplify the N-terminus region and primers O and J to amplify the C-terminal region. We mixed the two PCR products together and amplified the full- length intein with primers I and J. We cloned the PCR fragment into EcoRI/Xhol- ⁇ gested plN01 using in vivo homologous recombination in EY93 (Ma, H. et al. (1987) Gene 58:201-216).
  • pETIL-L2 We constructed pETIL-L2 by PCR amplifying the entire plL-L2 intein gene including the stop codon with primers P and Q. We digested the PCR fragment with EcoRI and Xhol (Fermentas) and cloned it into pET28b (Novagen).
  • pETIL-01 We constructed pETIL-01 by PCR amplifying the entire plN-01 intein gene including the stop codon using primers P and Q. We digested the PCR fragment with EcoRI and Xhol (Fermentas) and cloned it into pET28b (Novagen).
  • EY111 ::pEG202 cells were pelleted by centrifugation and resuspended the pellet in an equal volume of yeast peptone dextrose (YPD) media.
  • YPD yeast peptone dextrose
  • LB-KAN kanamycin
  • SMR6039 E. coli strain which expresses GFP under the control of a SOS- regulated sulA promoter (Hastings, PJ. et al. (2004) PLoS Biol. 2:e399) to monitor induction of the SOS response pathway.
  • ⁇ DE3 Lysog ⁇ nization Kit Novage ⁇
  • T7 RNA polymerase T7 RNA polymerase
  • MMC removed samples at specified time points, and diluted them in 2 mL 0.85 % NaCI for a final concentration of - 0.5 x 10 ⁇ cfu/mL.
  • MATCHING software was used to calculate the percentage of 16 O and 18 O incorporation in the two tryptic peptide fragments involved in the lactone bond, SWDLPGEY [966.42 m/z] [amino acids 73-80] and IFDIGLPQDHNFLLANGAIAHASR [2590.352 m/z][amino acids 49-72].
  • SWDLPGEY 966.42 m/z] [amino acids 73-80]
  • IFDIGLPQDHNFLLANGAIAHASR [2590.352 m/z][amino acids 49-72].
  • MATCHING software was used to determine the percentage of ' 6 O and 18 O incorporation assuming one oxygen incorporation.
  • MATCHING was used to determine the percentage of 18 O and 18 O incorporation assuming two oxygen incorporations.
  • the intensity of each peak is the sum of the maximum intensity of all peptides found in that peak multiplied by a scalar factor (I 1 ), which is the percentage of the maximum intensity of the peptide at that peak location predicted by MS-ISOTOPE software.
  • EQ2 was then simplified to a single variable by applying the constraints given by MATCHING software.
  • MATCHING software determined the ratio to be 14 % 16 O to 86% 18 O.
  • i is the number of different peptides in the model
  • j is the peak index
  • x ⁇ is the maximum intensity of a peptide
  • I j is a scale factor determined by MS-ISOTOPE for the fraction of x, expected at that peak location
  • lc f is the calculated peak intensity from the model at that peak location.
  • j is the peak index
  • lobSj is the intensity observed
  • lcalq is the calculated intensity
  • Amino acids in the extein at the intein-extein junction can effect splicing.
  • the Ssp DnaE intein has been shown to be promiscuous in regards to the amino acids that are found adjacent to the splice site. Mutation of wild-type inteins or using mixed inteins can alter this dependency. Iwai ⁇ tal., (Iwai, H. et al. (2006) FEBS Lett.
  • the plasm id backbone was modified to include a different selectable marker (Kan instead of Amp) as well as containing the I N domain of the Npu DnaE intein and the l c domain of the Ssp DnaE intein.
  • the L2 peptide (SRSWDLPGEY) isolated against LexA using the intein containing both domains from Ssp DnaE (Ic-Ssp, I N -SSP) was transferred to the (Ic-Ssp, I N - Npu) intein the L2 peptide still interacted with LexA in a yeast- two-hybrid assay and underwent processing.
  • Step 1 - 1 ⁇ g (20 ⁇ M) of oligonucleotides npu1 + npu2, npu3 + npu4, and npu5 + npuVR (FIg. 23), that have overlapping regions, were mixed together in separate PCR tubes with 60 rnM ThS-SO 4 (pH 8.9), 18 mM NH 4 SO 4 , 2 mM MgSO 4 , 10 mM dNTPs, and 1.0 Unit Platinum High Fidelity Taq (Invitrogen).
  • Step 2 full length Npu DnaE gene was constructed by mixing the dimers formed in Step 1 in a single reaction tube with 60 mM THs-SO 4 (pH 8.9), 18 mM NH 4 SO 4 , 2 mM MgSO 4 , 10 mM dNTPs, and 1.0 Unit Platinum High Fidelity Taq (Invitrogen) under the exact same conditions as in the dimer extension.
  • Step 3 the full length gene was selectively amplified from the pool of incomplete dimer extensions to result in the full length gene, 1 :10 of product from step (2) was mixed with npuVF, npuVR (FIg.23), 60 mM Tris-SO 4 (pH 8.9), 18 mM NH 4 SO 4 , 2 mM MgSO 4 , 10 mM dNTPs, and 1.0 Unit Platinum High Fidelity Taq (Invitrogen). The PCR reaction was initially denatured for 5 minutes at 95 0 C, followed by 25 cycles of 95 0 C for 30 seconds, 55 0 C for 30 seconds, and 72 0 C for 30 seconds.
  • the synthetic Npu DnaE gene was cloned into plN01 digested with Rsrll and Xhol (above) in the yeast strain EY93 by homologous recombination using lithium acetate transformations. This transformation resulted in the vector plUOO.
  • the Kan R gene was then cloned into plL.100 at the Amp R gene site.
  • plU OO was digested with Seal in NEBuffer 3 [100 mM NaCI, 50 mM Tris-HCI pH 7.9, 10 mM MgCI 2 , 1 mM Dithiothreitol] overnight at 37 0 C.
  • the Kan R gene was prepared by PCR amplification using S, T (Fig.
  • lactone-cyclized lariat The lariat peptide is generated by inhibiting Asn- cyclization in the intein-cyclization reaction, which produces a peptide that is cyclized through a lactone bond.
  • the lactone-bond cyclizing the lariat is more susceptible to hydrolysis than an amide bond and we have shown that - 50% of the lariat exists in the lactone-cyclized state when expressed in E. coli.
  • amino acids having other bulky side chains that possess an alkyl gamma carbon may be used stabilize the lactone (for example by blocking water from accessing the lactone bond).
  • the following amino acids may accordingly be substituted at position G7 (presented in order of preference for blocking water access to the lactone bond): Trp, Phe, Leu, lie, Tyr, Met, VaI, Arg, Lys, His, GIu 1 Asp
  • Asn at position lc-i is important for branched intermediate accumulation caused by lc-2 (G6) mutations.
  • mutations at position I 02 (G6) enhance lactone-cyclized lariat stability
  • Leu, Asn, and Asp mutations enhanced lariat stability to the 47%, 54%, and 55%, respectively.
  • the Leu mutation maintained good processing (72%), while the Asn and Asp mutations decreased processing to 19% and 8%, respectively.
  • amino acids having other hydrophobic side chains may also be used to stabilize lactone bond (for example by excluding water from the reactive site while still permitting processing). The following amino acids may also accordingly be substituted at position G6: Trp, Phe, Leu, He, Met, Tyr.
  • Position F4 (iv) Position F4 (Asp): The amino acid at this position coordinates water near the lactone bond and participates in Steps 1 and 2 by polarizing the carbonyl to assist in nucleophilic attack by Aland G8. Mutation of F4 from Asp to GIu, and GIn may accordingly be undertaken so as to allow Steps 1 and 2 to occur, while stabilizing the lactone bond (for example by excluding water from the region around the lactone bond).
  • Position F13 (His): A His to Ala mutation at F13 does not block Step 3, while substitution of a bulky hydrophobic amino acid at F13 may be used to stabilize the lactone bond: including substitution of Phe, Leu , or lie.
  • Position F14 (Asn): Bulky or charged amino acids substituted at F14 may be used to disrupt the correct positioning of F13 and thus block Asn cyclization and stabilize the lactone bond, including substitution of: Trp, Phe, Tyr, Leu, Lys, Arg
  • Position F15 (Phe): A mutation at F15 to Ala blocks Asn Cyclization, while mutation to Tyr slightly inhibits Asn cyclization. Accordingly, mutation of F15 to a bulky hydrophobic amino acid may be used to block Asn cyclization and exclude water around the lactone bond, thus stabilizing it. The following amino acids may accordingly be substituted at positioning of F13 to stabilize the lactone bond: Trp, Leu,
  • Mutations were constructed by site directed mutagenesis at the G6, G7, and B11 positions using Phusio ⁇ TM Site-Directed Mutagenesis Kit (Finnzymes) as per manufacturers instructions.
  • Phusio ⁇ TM Site-Directed Mutagenesis Kit (Finnzymes) as per manufacturers instructions.
  • We expressed the mutant L2 lariats by inducing a 0.6 OD 600 culture of BL21-CP with 1 mM IPTG for four hours.
  • ScFvs comprise immunoglobulin variable domains of heavy and light chains that are held together by a short peptide linker (Bird, R.E., et al. (1988) Science 242:423- 426).
  • Many ScFvs generated from natural antibodies or isolated by in vitro selection fail to function effectively in their designed application as they often denature or aggregate (Worn, A.
  • ScFvs are further destabilized by their inability to form a conserved i ⁇ tra-domain disulfide bond under the reducing conditions of the cytoplasm (Worn, A. & Pluckthun, A. (2001) J. MoI. Biol. 305:989-1010).
  • a variety of strategies, including rational and evolutionary approaches, have been used to enhance the intra- and inter-domain stability of ScFvs (Worn, A. & Pluckthun, A. (2001) J. MoI. Biol.
  • ScFv frameworks that are compatible with the yeast two-hybrid and other assays
  • ScFvs can be cyclized or their surface charge can be increased, both of which should enhance stability and solubility.
  • Crystal structures of ScFvs such as those reported by (Tanaka, T., et al., (2007) EMBO J. 26:3250-3259) can be used as guides for identifying surface residues to mutate.
  • Surface residues on the ScFv that are solvent accessible can be identified using ASAView software (Ahmad, S., et al., (2004) BMC Bioinf. 5:51-56) or other similar software and techniques for identifying surface residues.
  • Surface amino acids can be mutated to a positively (Lys, Arg, His) or negatively (Asp, GIu, Tyr, Cys) charged amino acids, depending on whether the desired charge on the ScFv is positive, negative, or a mixture of positive and negative charges.
  • ScFvs expressed as lariats, unprocessed, or dicystei ⁇ e inteins that interact with a given target can be isolated from synthetic ScFv libraries, where the variable regions or CDRs are randomized with two or more amino acids, using genetic assays such as the yeast two-hybrid assay. Once they are isolated, ScFvs can be stably produced by expressing them as lariats or as head to tail cyclized ScFvs using the intein-mediated cyclic peptide/pratein producing reaction. Alternatively, ScFvs can be cyclized by cross-linking. ScFvs can be engineered to contain small linker peptides at its N and C terminus that contain amino acids that can be can be cross-linked and give rise to a cyclized ScFv.
  • Cyclized and/or supercharged ScFvs for intracellular applications can also be constructed from existing monoclonal antibodies produced from hybridoma cell lines.
  • the heavy and light chain antibody cDNA is used as a template to PCR amplify the light and heavy chain variable domains. These domains can then be cloned into one of the describe intein expression constructs, where they will be translated as a lariat, unprocessed, or discysteine ScFvs.
  • the ScFv can be engineered to contain small linker peptides at its N and C terminus that contain amino acids that can be can be cross-linked and give rise to a cyclized ScFv.
  • ScFv and fragment antigen binding fragments that are isolated against a given target using an in vitro selection strategy such as phage display, yeast display, etc, can also be converted to an intracellular antibody by cyclizing and/or supercharging as described above.
  • the fragment antigen binding (Fab fragment) is a region on an antibody, which binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain.
  • Cyclization or supercharging can be also applied to the expression of heavy or light chain fragments alone.
  • the heavy and light chain are used as affinity agents alone. It is also possible the cyclized and/or supercharged heavy and light chains can be expressed separately and that they will interact and form a functional Fv composed of both chains.
  • Cydization or supercharging can be also applied to the expression of Fab fragments.
  • Fabs are composed of one constant and one variable domain of each of the heavy and the light chain. Light and heavy chain regions of Fabs are held together by an inter-domain disulfide bond. In this case, Fabs can be stabilized in a reducing environment such as is present inside cells, by cydization using one of the methods described above.
  • cyclized peptides, genomic fragments, and ScFvs can be delivered exogenously for in vitro or in vivo applications.
  • a variety of delivery systems are available for peptides using liposaccharides, lipopeptides, liposomes, and polyethylene glycol (PEG) conjugates (Reviewed by AIi, M & Manolios, N. (2002) Lett. Peptide Sci. 8:289-294).
  • Peptides and proteins can also be delivered by conjugating them, either covalently or non-covalently to transduction peptides (Reviewed by Joliot, A & Prochiantz, A. (2004) Nature Cell Biol. 6:189-196).
  • the eighteen oligonucleotides (Oligo1-Ab to Oligo18-Ab) were mixed together (0.2 ng/ ⁇ L of each) with HIFI Taq polymerase Buffer (60 mM TnS-SO 4 (pH 8.9), 18 mM (NH 4 ) 2 SO4) (I ⁇ vrtrogen), 0.2 mM dNTPs, 2 mM MgSO 4 , and1.0 Unit Platinum HIFI Taq polymerase (Invitrogen).
  • the reaction mix was incubated under the following conditions: 94 0 C for 2 minutes, (94 0 C for 30 seconds, 56 0 C for 30 seconds, 68 0 C for 1 minute (30 cycles), and 68 0 C for 10 minutes.
  • ScFv framework into yeast expression vector plL500 was digested with 0.5 Units EcoRI endonuclease, 1 Units Xhol endonuclease, 1X y+/Tango Buffer (Fermentas) to remove the intei ⁇ sequence. The reaction mixture was incubated at 37 0 C overnight. The ScFv framework was cloned into EcoRI and Xhol digested plL500 using homologous recombination. EcoRI and Xhol digested plL500 and 40 ⁇ L of PCR amplified ScFv framework were transformed into yeast strain EY93 as described by Gietz et al. (Schiestl, R. H. & Gietz, R. D. (1989). Curr. Genet. 16:339-346) giving rise to the ScFv framework expression plasmid referred to as pScFv-Fr
  • CDR Library Oligonucleotides The CDRs were randomized by cloning degenerate oligonucleotides flanked by fixed regions into the ScFv framework using homologous recombination.
  • T4 libraries contain combinatorial Tyr and Ser CDRs in heavy chain CDRs 1-3 and light chain CDR3.
  • K4 libraries contain combinatorial Tyr, Ser, Ala, and Asp CDRs in heavy chain CDRs 1-3 and light chain CDR3.
  • Oligonucleotides containing the degenerate CDRs are listed in Figure. 28.
  • pScFv-Fr plasmid was digested with Xhol and gel purified.
  • the degenerate CDR3 regions for the K or T libraries were cloned into pScFv-Fr using homologous recombination by transforming Xhol digested pScFv-Fr and PCR amplified Oligo-CDR3KMT into EY93 using lithium acetate transformation (Schiestl, R. H. & Gietz, R. D. (1989) Curr. Genet.
  • Oligo-CDR3KMT was PCR amplified in the following reaction: 1X PCR Buffer, 0.2 mM dNTPs, 2 mM MgSO 4 , 1 ⁇ M CDR.FWD, 1 ⁇ M CDR3.RVS, 0.02 ⁇ M Oligo-CDR3KMT or Oligo-CDR3KMT, 72 ⁇ l_ H 2 O, 0.4 ⁇ l_ Taq polymerase.
  • the PCR reaction was incubated under the following conditions: 95 °C for 1 minute, 95 ' C for 30 seconds, 52 ' C for 30 seconds, and 68 °C for 30 seconds (20 cycles).
  • pScFv-Fr-HCD3-K and pScFv-Fr-HCD3-T was digested with Nrul and gel purified.
  • CDRs land 2 were PCR amplified in the following reaction: 1X PCR Buffer, 0.2 mM dNTPs, 2 mM MgSO 4 , 0.02 ⁇ M Oligo-CDR1TMT (or KMT), 0.02 ⁇ M Oligo-CDR2 TMT (or KMT), 0.2 ⁇ M CDR1-F2-CDR2.RVS, 0.2 ⁇ M CDR1-F2-CDR2.FWD, 80 ⁇ l_ H 2 O, 0.4 ⁇ L Taq Polymerase.
  • the PCR reaction was incubated under the following conditions: 95 ° C for 2 minutes, 95 'C for 30 seconds, 56 'C for 30 seconds, 68 'C for 1 minute (25 cycles) and 68' C for 10 minutes.
  • the degenerate K or T CDR1 and 2 regions were cloned into pScFv-Fr-HCD3-K and pScFv-Fr-HCD3-T, respectively using homologous recombination by transforming Nrul digested pScFv-Fr-HCD3-K or pScFv-Fr-HCD3-T and PCR amplified CDR1 and 2 into EY93 using lithium acetate transformation (Schiestl, R. H. & Gietz, R. D. (1989) Curr. Genet. 16:339-346), which gives the new plasmid pScFv-Fr-HCD1-3-K and pScFv-Fr- HCD1-3-T.
  • PCR reaction conditions 1X PCR Buffer (InvUrogen), 0.2 mM dNTPs, 1 ⁇ l_ pScFv-Fr-HCD1-3-K or pScFv-Fr-HCD1- 3-T, 0.6 ⁇ M P1 pJG4-5 ChK, 0.2 ⁇ M TMT (or KMT)L3.RVS, 0.2 ⁇ M Ab33.pJG26.RVS, 0.2 ⁇ M pJG4-5.RVS, 1 ⁇ L Taq polymerase.
  • the reaction mixture was incubated under the following conditions: 95 0 C for 2 minutes, 95 0 C for 30 seconds, 55 0 C for 30 seconds, 72 0 C for 30 seconds (25 cycles), and 72 "C for 10 minutes.
  • the PCR product was cloned into plL500 digested with EcoRl and Xhol, giving rise to the plasmids expressing the K4 and T4 libraries, referred to as pScFv-K4 or pScFv-T4.
  • Cyclization of ScFv Library plL.500 was digested with 10 Units of Nru ⁇ and 1X NEBuffer in a 100 ⁇ L reaction. The reaction was incubated at 37 0 C for 24 hours.
  • DNA encoding ScFvs with T4 or K4 libraries were amplified from pScFv-K4 or pScFv-T4 using PCR using primer P1 VH3-74/plL500 ⁇ Fig. 28), which contain overlapping complementary sequences to the IC domain.
  • the second primer (P2 L19/Linker) (FIg. 28) contains DNA encoding a second linker peptide, which adds a peptide linker between the V H domain and the I N domain.
  • ScFv libraries were PCR amplified using the following conditions: 1 X PCR Buffer, 0.2 mM dNTPs, 1 ⁇ L pScFv-K4 or pScFv-T4, 0.2 ⁇ M P1 VH3-74/plL500, 0.2 ⁇ M P2 L19/Linker, and 1 ⁇ L Taq polymerase.
  • the PCR reaction was incubated under the following conditions: 95 0 C for 2 minutes, 95 0 C for 30 seconds, 50 0 C for 30 seconds, 72 0 C for 2 minute (30 cycles), and 72 0 C for 7 minutes.
  • a second PCR reaction was performed using a primer (P2 Unker/plL ⁇ OO) that adds DNA that overlaps sequences to the IN domain.
  • the reaction was performed as described above.
  • the PCR product was cloned into plLSOO digested with NmI giving rise to the plasmids expressing the K4 and T4 libraries, referred to as pScFv- cyc-K4 or pScFv-cyc-T4.
  • 50 members from each library were sequenced to determine the percentage of functional ScFvs and to confirm library diversity.
  • K4, cyc-K4, T4 and cyc-T4 libraries were screened against a pool of five baits: Bcr-Abl SH2 Domain, Bcr-Abl SH3 Domain, Bcr-Abl Coiled-coil domain, Bcr-Abl Y177 Motif, and Hck Tyr Kinase Domain.
  • T4, Cyc-T4, K4, and cyc-K4 libraries were transformed into E ⁇ Y93 to give a final library diversity of 4.2x10 ⁇ , 4.2 x10 ⁇ , 20 x10 6 , and 2.2 x10 6 , respectively.
  • ScFv libraries and bait cells were cultured overnight in Trp- Glucose and His- Glucose media, respectively, to an optical density above 0.5. Cells were centrifuged at 4000 rpm for 5 minutes at room temperature and washed in 1X PBS. The cells were centrifuged again as above and re-suspended in YPD + Adenine (40 mg/L) media. Cells were mixed at a 60x10 ⁇ ScFv library cells to 30x10 ⁇ of each bait (Total baits 150x10 e ) ratio and plated on YPD + Adenine plates and incubated overnight at 30 ° C.
  • the cells were re-suspended in 100 ⁇ L H 2 O and 100 ⁇ L 0.2 M NaOH, lightly vortexed, and incubated for 5 minutes at room temperature.
  • the reaction was centrifuged and re- suspended in 50 ⁇ L SDS-loading buffer (0.06 M Tris-HCI, pH 6.8, 5% glycerol, 2% SDS 1 4% ⁇ -mercapto-ethanol, 0.0025% bromophenol blue) and heated for 3 minutes at 95 1 C.
  • the samples were analyzed using 15 % SDS PAGE.
  • the gel was electrablotted to a nitrocellulose membrane for 45 minutes at 15 V.
  • the nitrocellulose membrane was incubated in 10 mL blocking buffer (Licor Biosciences) for one hour.
  • the membrane was incubated in an ⁇ -HA primary antibody solution (50 ⁇ L of ct-HA antibody (Santa Cruz), 10 mL blocking buffer, 5 ⁇ L Tween) overnight.
  • the membrane was washed three times with 1X PBS incubated for one hour with ⁇ -mouse secondary antibody (Licor Biosciences).
  • the membrane was washed 3 times with 1 X PBS and visualized using infrared Licor Analyzer.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte dans ses divers aspects sur des méthodes de cyclisation de protéines y compris des méthodes augmentant la stabilité de protéines cyclisées dans des conditions cytosoliques. L'invention porte également sur diverses méthodes d'utilisation de protéines cyclisées qui peuvent par exemple servir dans des essais de criblage analogues aux essais à double hybride de levure. Certaines exécutions sélectionnées de l'invention portent sur des molécules cyclisées de fragments variables à chaîne simple (ScFv), dont des molécules d'immunoglobuline repliée.
PCT/CA2008/000048 2007-01-10 2008-01-10 Stabilisation de structures de peptides cycliques WO2008083493A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002675024A CA2675024A1 (fr) 2007-01-10 2008-01-10 Stabilisation de structures de peptides cycliques
US12/522,708 US20130273553A9 (en) 2007-01-10 2008-01-10 Stabilization of cyclic peptide structures
EP08700513A EP2106446A4 (fr) 2007-01-10 2008-01-10 Stabilisation de structures de peptides cycliques

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US88427907P 2007-01-10 2007-01-10
US60/884,279 2007-01-10
US60/844,279 2007-01-10

Publications (2)

Publication Number Publication Date
WO2008083493A1 true WO2008083493A1 (fr) 2008-07-17
WO2008083493A8 WO2008083493A8 (fr) 2013-10-24

Family

ID=39608298

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2008/000048 WO2008083493A1 (fr) 2007-01-10 2008-01-10 Stabilisation de structures de peptides cycliques

Country Status (5)

Country Link
US (1) US20130273553A9 (fr)
EP (1) EP2106446A4 (fr)
CN (1) CN101646771A (fr)
CA (1) CA2675024A1 (fr)
WO (1) WO2008083493A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107236020A (zh) * 2017-06-14 2017-10-10 东华大学 一种同一体系中同时对两个蛋白进行特异性标记或修饰的方法
AU2018223040B2 (en) * 2014-12-05 2020-04-30 Xyphos Biosciences Inc. Insertable variable fragments of antibodies and modified a1-a2 domains of NKG2D ligands
WO2020123387A1 (fr) * 2018-12-10 2020-06-18 Lassogen, Inc. Systèmes et procédés de découverte et d'optimisation de peptides lasso
EP3828200A4 (fr) * 2018-07-09 2022-05-18 National University Corporation Kumamoto University Anticorps à chaîne unique cyclique
EP3994251A4 (fr) * 2019-07-01 2023-09-20 The Governing Council of the University of Toronto Détection d'interactions protéine-protéine

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2468881A3 (fr) * 2005-07-21 2012-08-15 Abbott Laboratories Expression de gènes multiples incluant des constructions molles avec des polyprotéines, des pro-polyprotéines et protéolyse
JP2013509188A (ja) * 2009-10-30 2013-03-14 アボット・ラボラトリーズ Ｓｏｒｆ構築物および複数の遺伝子発現
CN101812449A (zh) * 2010-04-06 2010-08-25 东华大学 一种小环肽的制备方法
US8962550B2 (en) 2010-12-17 2015-02-24 The Penn State Research Foundation Identification of inhibitors of a bacterial stress response
CN106749497B (zh) * 2017-02-06 2020-10-30 陈铭 套索多肽的化学全合成
US20200102356A1 (en) * 2018-10-01 2020-04-02 NMC Inc. Compositions and Methods for the Treatment of Huanglongbing (HLB) aka Citrus Greening in Citrus Plants

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036093A2 (fr) * 1998-12-18 2000-06-22 The Penn State Research Foundation Cyclisation de peptides a mediation inteine

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004030610A2 (fr) * 2002-07-11 2004-04-15 The Trustees Of Columbia University In The City Of New York Compositions et procedes pour la delivrance intracellulaire d'anticorps
EP1811031A1 (fr) * 2006-01-18 2007-07-25 Millegen Méthode de sélection d'un peptide ou polypeptide capable de se fixer à une molécule cible

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000036093A2 (fr) * 1998-12-18 2000-06-22 The Penn State Research Foundation Cyclisation de peptides a mediation inteine

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
COOPER A.A. ET AL.: "Protein splicing of the yeast TFP1 intervening protein sequence: a model for self-excision", EMBO J., vol. 12, no. 6, June 1993 (1993-06-01), pages 2575 - 2583, XP002105837 *
DING Y. ET AL.: "Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing", J. BIOL. CHEM., vol. 278, no. 40, 3 October 2003 (2003-10-03), pages 39133 - 39142, XP008111031 *
KAWASAKI M. ET AL.: "Identification of three core regions essential for protein splicing of the yeast Vma1 protozyme", J. BIOL. CHEM., vol. 272, no. 25, June 1997 (1997-06-01), pages 15668 - 15674, XP008111032 *
PERLER F.B. ET AL.: "Compilation and analysis of intein sequences", NUC. ACIDS RES., vol. 25, no. 6, March 1997 (1997-03-01), pages 1087 - 1093, XP002154590 *
See also references of EP2106446A4 *
XU M.-Q. AND PERLER F.B.: "The mechanism of protein splicing and its modulation by mutation", EMBO J., vol. 15, no. 19, October 1996 (1996-10-01), pages 5146 - 5153, XP001063196 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2018223040B2 (en) * 2014-12-05 2020-04-30 Xyphos Biosciences Inc. Insertable variable fragments of antibodies and modified a1-a2 domains of NKG2D ligands
CN107236020A (zh) * 2017-06-14 2017-10-10 东华大学 一种同一体系中同时对两个蛋白进行特异性标记或修饰的方法
EP3828200A4 (fr) * 2018-07-09 2022-05-18 National University Corporation Kumamoto University Anticorps à chaîne unique cyclique
WO2020123387A1 (fr) * 2018-12-10 2020-06-18 Lassogen, Inc. Systèmes et procédés de découverte et d'optimisation de peptides lasso
EP3994251A4 (fr) * 2019-07-01 2023-09-20 The Governing Council of the University of Toronto Détection d'interactions protéine-protéine

Also Published As

Publication number Publication date
CN101646771A (zh) 2010-02-10
CA2675024A1 (fr) 2008-07-17
US20130273553A9 (en) 2013-10-17
EP2106446A1 (fr) 2009-10-07
EP2106446A4 (fr) 2010-06-16
WO2008083493A8 (fr) 2013-10-24
US20100129807A1 (en) 2010-05-27

Similar Documents

Publication Publication Date Title
US20100129807A1 (en) Stabilization of cyclic peptide structures
US20180017573A1 (en) Polynucleotide construct capable of displaying fab in a cell-free translation system, and method for manufacturing and screening fab using same
KR101796689B1 (ko) 안정화된 면역글로불린가변도메인 선별방법 및 선별된 도메인의 응용
EP3066120B1 (fr) Anticorps à domaine unique synthétique
JP2009520468A (ja) 融合タンパク質ライブラリーの作製法およびスクリーニング法、ならびにそれらの使用
CA2681170C (fr) Procedes de production d'anticorps scfv actifs et bibliotheques de ceux-ci
EP3882348A2 (fr) Intracorps ciblant des modifications post-translationnelles de protéines natives et leur procédé d'obtention
ES2819865T3 (es) Proteínas dirigidas a ortólogos
US20210032368A1 (en) Methods and compositions for the development of antibodies specific to epitope post-translational modification status
Nishi et al. Ligation-based assembly for constructing mouse synthetic scFv libraries by chain shuffling with in vivo-amplified VH and VL fragments
US11339388B2 (en) Methods and compositions for generation of binding agents against cell surface antigens
Barreto A genetic screen to isolate “lariat” peptide inhibitors of protein function
KR20180104675A (ko) 합성 항체 라이브러리의 생성 방법, 상기 라이브러리 및 그 적용(들)
AU2015202410B2 (en) Immunoglobulin frameworks which demonstrate enhanced stability in the intracellular environment and methods of identifying same
da Silva Recombinant Intracellular Antibodies For Molecular Gene Therapy of HIV-1 Infection

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200880007136.8

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08700513

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2675024

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2008700513

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 12522708

Country of ref document: US